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Article Cite This: J. Med. Chem. 2018, 61, 7814−7826

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Total Synthesis and Structure−Activity Relationships Study of Odilorhabdins, a New Class of Peptides Showing Potent Antibacterial Activity Matthieu Sarciaux,† Lucile Pantel,† Camille Midrier,‡ Marine Serri,† Cristelle Gerber,† Renata Marcia de Figueiredo,‡ Jean-Marc Campagne,‡ Philippe Villain-Guillot,† Maxime Gualtieri,† and Emilie Racine*,† Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on September 19, 2018 at 18:21:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Nosopharm, 110 allée Charles Babbage, Espace Innovation 2, 30000 Nîmes, France Institut Charles Gerhardt Montpellier (ICGM), UMR 5253, Univ Montpellier, CNRS, ENSCM - Ecole Nationale Supérieure de Chimie, 8 Rue de l’Ecole Normale, Montpellier 34296 Cedex 5, France

‡

S Supporting Information *

ABSTRACT: The spread of antibiotic-resistant pathogens is a growing concern, and new families of antibacterials are desperately needed. Odilorhabdins are a new class of antibacterial compounds that bind to the bacterial ribosome and kill bacteria through inhibition of the translation. NOSO-95C, one of the first member of this family, was synthesized for the first time, and then a structure−activity relationships study was performed to understand which groups are important for antibacterial activity and for inhibition of the bacterial translation. Based on this study an analogue showing improved properties compared to the parent compound was identified and showed promising in vitro and in vivo efficacy against Enterobacteriaceae.

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antibacterials.11 Recently Gram-negative bacteria have attracted interest for antibiotic discovery and have proven to be an interesting source of new active structures.12,13 In this line, the antibacterial screening of a collection of Xenorhabdus strains by our team has led to the discovery of the Odilorhabdins (ODLs), a novel antibiotic class with a broadspectrum activity against Gram-positive and Gram-negative pathogens.14−16 ODLs are a family of peptides that inhibit specifically the bacterial translation with a novel mode of action by interacting with the 30S subunit of the bacterial ribosome. Further evaluation of this new family of peptides has shown promising in vitro antibacterial efficacy against a panel of resistant bacteria, including CRE, and engaging in vivo efficacy in infectious models with Gram-positive and Gramnegative bacteria.15 In parallel, no cytotoxicity was observed at concentrations 30- to 100- fold higher than concentrations needed to observe antibacterial effect against Enterobacteriaceae, confirming the great potential of this new family. Based on these promising results, a medicinal chemistry program was engaged to study the structure−activity relationships (SAR) of these peptides to understand better which amino acids are important for antibacterial activity and for inhibition of the bacterial translation. NOSO-95C 1 was one of

INTRODUCTION The emergence of antimicrobial resistance (AMR) to the major classes of antibacterial drugs is a significant public health problem as highlighted recently in the World Health Organization’s (WHO) Global Antimicrobial Surveillance System (GLASS) report.1 Among resistant bacteria, carbapenem resistant Enterobacteriaceae (CRE) are one of the biggest threats.2,3 Different mechanisms may contribute to render these bacteria resistant to carbapenems, but the most important clinically is the production of various classes of carbapenemases (like KPC, VIM-, IMP-, NDM-, or OXAtypes) that hydrolyze almost all β-lactams, confer high levels of carbapenem resistance, and are commonly associated with genes encoding for resistance to other classes of antibacterials.4 Currently none of the novel combinations of β-lactams with βlactamase inhibitors or new compounds derived from old antibacterial classes are effective against all classes of carbapenemases.5 Based on these alarming data, recent reports from WHO have classified the R&D of new antibacterials to fight CRE at the highest level of priority and have highlighted the need for new structures and mechanisms of action.6,7 As opposed to target-based drug discovery which has been disappointing for the discovery of new antibacterial structures,8 natural products isolated from bacteria and fungi have been the main source of clinically used antibiotics9,10 and are still a promising source for the discovery of new families of © 2018 American Chemical Society

Received: May 17, 2018 Published: August 7, 2018 7814

DOI: 10.1021/acs.jmedchem.8b00790 J. Med. Chem. 2018, 61, 7814−7826

Journal of Medicinal Chemistry

Article

Figure 1. Structure of NOSO-95C 1.

Scheme 1. Synthesis of Protected Dab(βOH) 6a

the first peptide isolated from the antibacterial screening and was selected as the starting point for this study (Figure 1). It is a 10-mer linear cationic peptide containing six proteinogenic and four nonstandard amino acids: ((2S,3S)-α,γ-diamino βhydroxy butyric acid (Dab(βOH)) in positions 2 and 3, Dornithine (D-Orn) in position 5, Z-α,β−dehydroarginine (Dha) in position 9, and a functionalized secondary amide at the Cterminal position (α,δ−diamino butane (Dbt)). In this paper we report on the first synthesis of NOSO-95C after having developed the synthesis of the two nonstandard amino acids Dab(βOH) and Dha. Once the structure of the peptide validated a SAR study was initiated to identify the moieties involved in the antibacterial efficacy and in the inhibition of the bacterial translation. Interestingly, a promising simplified analogue was identified and showed improved characteristics compared to the natural compound.

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RESULTS AND DISCUSSION Synthesis of NOSO-95C. The synthesis of NOSO-95C 1 was planned via solid phase peptide synthesis (SPPS) using a Fmoc-strategy.17,18 This highly efficient technique requires the use of amino acids bearing appropriate protecting groups which were commercially available for all building-blocks except for the two original amino acids Dab(β−OH) and Dha. Thus, the synthesis of these two protected amino acids was first developed. Due to the large amount of each building block required for the production of numerous analogues, the syntheses were designed to be short, efficient, cheap, and scalable. Moreover, a high chiral purity would have to be obtained as separation of diastereoisomers once the peptide is obtained could be very challenging. The synthesis of Dab(βOH) or its protected analogues has already been described, but the high number of steps and/or the low chiral purity of the product obtained make them not suitable for multigram scale synthesis.19−23 (4S,5S)-5Hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidino-4-carboxylic acid 2 (5-hydroxyectoin, Scheme 1) is produced by many halophilic microorganisms and is commercially available and affordable. 24 Its structural similarity with Dab(βOH), especially regarding the stereochemistry of chiral centers, makes it a suitable starting material for the synthesis of a protected Dab(βOH). The synthesis of 6 from 5-hydroxyectoin was thus investigated (Scheme 1).25 Opening of the six-membered ring was achieved using two equivalents of NaOH. The monoacetylated intermediate (not isolated) was then treated in acidic conditions to give unprotected Dab(βOH) 3. Marfey’s analysis of the crude mixture showed a 3:1 ratio between 2S,3S and 2S,3R

a Reagents and conditions: (a) NaOH (2.0 equiv), H2O, 50 °C, 6 h. (b) HCl 6 N, 110 °C, 3 h. (c) NaOH (3.0 equiv), H2O, CuSO4·5H2O (0.5 equiv), 25 to 110 °C then rt, 5 h. (d) Boc2O (2.0 equiv), dioxane, rt, 72 h then Boc2O (0.5 equiv), dioxane, rt, 24 h. (e) NaOH (3.0 equiv), Na2EDTA·2H2O (1.5 equiv), H2O, rt, 4 h. (f) Fmoc-OSu (2.5 equiv), Na2CO3 (2.5 equiv), dioxane, 0 °C then rt, 18 h. (g) Acetone:2,2-dimethoxypropane (1:1), BF3·OEt2 (cat.), 0 °C to rt, 2 h.

diastereoisomers, respectively.26 Attempts to improve this ratio were unsuccessful (unpublished data), and the mixture of isomers was sent to the next step without further purification. Suitable protections were introduced following a protocol described for (5-OH)-lysine. 27 First the α-amino and carboxylate functions were regioselectively protected through formation of a copper-complex in basic conditions.28−30 The free amine of the corresponding copper complex was then protected by reaction with Boc2O and afforded compound 4 with a 40% yield over 4 steps. Decomplexation of the copper was achieved by treating 4 with EDTA and was followed by protection of the α-amino function using Fmoc-OSu. The crude oil obtained was precipitated with acetonitrile, and to our great surprise only the expected 2S,3S diastereoisomer 5 was obtained.31 Finally protection of the amino alcohol was achieved by dissolving 5 in a 1:1 mixture of acetone and 2,2dimethoxypropane and treating the solution with BF3·OEt2. The protected Dab(βOH) 6 was obtained with a 73% yield and a diastereoisomeric ratio higher than 95:5 (based on Marfey’s analysis). 7815



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3). Aldehyde 15 was obtained with an excellent yield and was used within few days in the next step without further purification due to its instability.

Regarding the synthesis of the dehydroarginine (Dha) a different strategy was investigated. Dehydroamino acids (Dhaa) are well-known to be poor nucleophiles in peptide coupling reactions due to the delocalization of the nitrogen lone pair.32 Moreover, their free amino form is unstable, readily hydrolyzing in mildly acidic conditions to liberate ammonia and an α-keto acid.33 In contrast, N-acylated Dhaa are stable and are hydrolyzed only under more rigorous conditions. Based on this, synthetic routes involving the use of simple Dhaa should be avoided and the synthesis of a dipeptide where the amino function is involved in an amide bond with the next amino acid should be favored.34,35 Therefore, the synthesis of a protected dipeptide lysine-dehydroarginine 7 was investigated (Figure 2). Formation of the double bond was planned via a Horner−Wadsworth−Emmons reaction, a method first described by Schmidt et al.36 and successfully scaled-up by Bayer Pharma.37

Scheme 3. Synthesis of Aldehyde 15a

a

Reagents and conditions: (a) 3-amino-propan-1-ol (4.0 equiv), DMAP (0.1 equiv), DMF, rt, 4 h. (b) Dess−Martin periodinane (1.1 equiv), pyridine (6.0 equiv), DCM, rt, 3 h.

Horner−Wadsworth−Emmons olefination was set up between phosphonate 12 and aldehyde 15 using modified Masamune−Roush conditions in order to obtain a suitable Z/ E ratio (Scheme 4).38 A 86/14 ratio between Z and E isomers was obtained in the crude mixture, and the Z isomer was finally obtained with a 71% yield and a >95/5 Z/E ratio after purification by column chromatography. 1H NMR analysis (NOESY) showed a clear interaction between the NH (9.27 ppm) and the χCH2 of the dehydroarginine (2.39 ppm) while no interaction was observed between this same NH and the vinylic CH (6.46 ppm). The E isomer was also isolated, and in this case, NOESY analysis showed a clear interaction between the NH of the dehydroarginine (9.52 ppm) and the vinylic CH (5.93 ppm) while no interaction was observed between this same NH and the χCH2 of the dehydroarginine (2.54 ppm). Saponification of the ester function led to the dipeptide (Z)-7 with a modest yield due to partial cleavage of the Fmoc function, despite the presence of CaCl2 in the reaction mixture.39 Attempts to improve this step (decrease the temperature, Fmoc reprotection of the crude mixture, saponification using AlCl3/dimethylaniline40) were unsuccessful. With these two building-blocks in hand, the synthesis of NOSO-95C 1 was investigated via solid-phase peptide synthesis (SPPS)17 using the orthogonal Fmoc/tBu strategy.41,42

Figure 2. Structure of the dipeptide Lys-Dha 7.

Condensation between glyoxylic acid 8 and benzyl carbamate led to the amino acid 9 with a good yield (Scheme 2). Esterification of the acid and etherification of the hydroxyl function led to the ester 10. Transformation of the ether function into a chloride followed by substitution by triethyl phosphite was made in one pot to yield phosphonate 11. The amine function was deprotected by hydrogenation and the free amine intermediate was immediately reacted with FmocLys(Boc)−OH to provide the protected amino acid 12, first intermediate of this synthesis. The second building-block was obtained by reacting 3amino propan-1-ol and thiourea 13 to give alcohol 14 which was then oxidized using Dess−Martin periodinane (Scheme Scheme 2. Synthesis of Phosphonate 12a

Reagents and conditions: (a) Cbz-NH2 (0.9 equiv), toluene, 40 °C, 1.5 h. (b) HCl (cat.), trimethyl orthoformate (2.0 equiv), MeOH, 56 °C, 40 min. (c) H2SO4 (cat.), PCl3 (1.2 equiv), toluene, 75 °C, 13 h then P(OEt)3 (1.1 equiv), 75 °C, 2 h then 90 °C, 30 min (d) (1) Pd/C 10% (10 wt %/wt), H2, EtOH, rt, 14 h. (2) Fmoc-Lys(Boc)−OH (1.0 equiv), PyBOP (1.0 equiv), DCM, 0 °C then DIPEA (3.0 equiv), rt, 2 h.

a

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Scheme 4. Synthesis of Dipeptide 7a

a Reagents and conditions: (a) phosphonate 12 (1.0 equiv), LiCl (1.2 equiv), AcCN, rt, 30 min then aldehyde 15 (1.2 equiv), DIPEA (1.2 equiv), rt, 4 days. (b) CaCl2 0.8 M (13.0 equiv), iPrOH/H2O (7:3), rt, 20 min then aq. NaOH 1 M (2.0 equiv), 0 °C to rt, 16 h.

The synthesis was initiated through coupling the first amino acid, Fmoc-Lys(Boc)−OH, on the commercially available 1,4diaminobutyl 2-chlorotrityl chloride resin by using the uronium reagent HBTU (O-(benzotriazol-1-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate) in DMF/ DIPEA.43,44 Subsequent elongation of the peptide was achieved through stepwise coupling of the following Fmocamino acids, namely Fmoc-Lys(Boc)-Dha(Boc)2−OH 7, Fmoc-His(Trt)−OH, Fmoc-D-Orn(Boc)−OH, Fmoc-GlyOH, protected Dab(βOH) 6 twice, and Fmoc-Lys(Boc)− OH. At the end of each coupling step, Fmoc deprotection was carried out using a solution of 20% piperidine in DMF. At the end of the synthesis, acid sensitive lateral chains’ deprotections and cleavage of the peptide from the resin were made in one step using a solution of TFA, water, and triisopropylsilane (TIS) as scavenger. Purification of the deprotected peptide was achieved through reverse phase column chromatography to give the expected NOSO-95C 1 as a TFA salt showing a purity of 96.5% (LC-MS). Stereochemistry of the double bond was checked with 1H NMR (ROESY) and a clear correlation between NH of the Dha (9.70 ppm) and χCH2 of the Dha (2.28 ppm) was observed confirming the Z stereochemistry (see the Supporting Information (SI), Figure 31). No correlation was observed between NH of the Dha (9.70 ppm) and the vinylic CH (6.27 ppm). Chiral purity was measured using the Marfey’s analysis and unwanted diastereoisomers could not be detected (see the SI, Table S2).27 1H NMR and LC-MS spectra of the natural and synthetic peptide were found to be similar (see the SI, Figures 25−30 and Table S1). Finally, in vitro antibacterial activity against Enterobacteriaceae was compared by using minimal inhibitory concentration (MIC) assays45 on wild-type strains of E. coli and K. pneumoniae, the most problematic bacterial species in term of incidence of hospital-acquired infections (Table 1).46 The same efficacy was observed for both peptides. With a synthetic method in hand for this family of peptides, a SAR study was started with the aim of understanding better which parts of the molecule are crucial for antibacterial activity and for inhibition of the bacterial translation.

Structure−Activity Relationships on NOSO-95C. All of the analogues were synthesized via SPPS following the method described previously. NOSO-95C 1 and novel analogues were evaluated for in vitro antibacterial activity in minimal inhibitory concentration (MIC) assays against two wild-type Enterobacteriaceae: E. coli ATCC 25922 and K. pneumoniae ATCC 43816.46 In antibacterial research, the MIC determination assay is the standard test to characterize the in vitro potential of a compound, but MICs values are the result of variables such as compound permeability across membranes and target effects. Thus, IC50 of the inhibition of the bacterial translation was measured using an in vitro coupled transcription-translation (IVTT) cell extract assay to confirm that the analogues inhibited protein synthesis and to determine whether cellular penetration was affecting antibacterial activity. Before starting the SAR study, IC50 of synthetic NOSO-95C was measured at 0.63 μM. This IC50 value is in the range of other translation inhibitors like gentamicin (0.31 μM), chloramphenicol (7.74 μM), spectinomycin (0.99 μM), or erythromycin (2.12 μM). The first step of this SAR study was to run an alanine scanning (Ala scan), a well-known and powerful method for studying the impact of lateral chains on the activity of peptides (Table 2).47,48 Modification of the lateral chain of noncommercial amino acids Dab(βOH) and Dha was also investigated. Finally, the removal of amino acids from N- or C-terminal positions of the peptide was tested to find the shorter active sequence. Analogues bearing an alanine at each amino acid position were prepared and evaluated. Removal of the lateral chain of Dab(βOH)2 (18) and D-Orn5 (21) led to a drastic decrease of the antibacterial activity on both strains and a loss of activity in the IVTT assay, especially for D-Orn5. As shown later on during a cocrystallization study between a shorter analogue and the bacterial ribosome the lateral chains of Dab(βOH)2 and DOrn5 interact directly with the ribosome and are key for the inhibition of translation.14 Modification of Dab(βOH)2 was further investigated. First the influence of the hydroxyl group was studied by introducing 2,4-diaminobutyric acid (Dab) in this position (28). A strong decrease of the antibacterial activity on both strains and a 10-fold decrease of the inhibition of translation were observed. Influence of the amine function was then studied by replacing Dab(βOH) by allo-threonine (AlloThr, 29) or Ser (30). Again, loss of the antibacterial activity and a decrease of the inhibition of the translation even stronger than in the case of Dab (28) were observed. These results confirm the high importance of Dab(βOH)2, especially of its amine function, for antibacterial activity. When Lys1 (17), His7 (23), Lys8 (24), and Lys10 (26) were replaced by an alanine, antibacterial activity on E. coli was

Table 1. In Vitro Antibacterial Activity of Synthetic versus Natural NOSO-95C 1a MIC (μg/mL) compound

E.c. ATCC 25922

K.p. ATCC 43816

NOSO-95C (natural) NOSO-95C (synthetic)

8 8

4 4

a

Abbreviations are as follows: MIC: minimum inhibitory concentration; E.c.: Escherichia coli, K.p.: Klebsiella pneumoniae. 7817



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Table 2. Ala Scan and Modifications of Lateral Chains of NOSO-95C 1a peptide sequence cmpd 1 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

K

Dβo

Dβo

G

D-Orn

P

H

MIC (μg/mL) K

dhR

K

Dbt

A A A A A A A A A A CO2H Dab Tal S Dab Tal S G V Abu dhAbu R r

E.c.

K.p.

8 16 >64 2 >64 >64 16 8 16 >64 8 16 >64 >64 >64 4 8 4 8 4 4 16 32 16

4 8 >64 1 >64 >64 32 8 4 64 8 4 64 >64 >64 4 8 4 8 2 4 8 8 16

IC50 (μM, ± SD) 0.63 ± 0.73 ± 4.50b 0.62 ± 3.64 ± >40 2.41 ± 0.39 ± 1.85 ± 2.40 ± 1.04 ± 0.36 ± 5.91 ± >40 >40 1.10 ± 1.00 ± 0.66 ± 1.06 ± 0.80 ± 0.73 ± 0.83b 1.53 ± 0.98 ±

0.05 0.05 0.05 0.01 0.06 0.02 0.16 0.34 0.28 0.08 0.34

0.39 0.06 0.03 0.04 0.02 0.04 0.06 0.05

a Abbreviations are as follows: Cmpd: compound; MIC: minimum inhibitory concentration; Dβo: Dab(βOH); dh: dehydro; Dbt: 1,4-diamino butane; Dab: 2,4-diaminobutyric acid; Abu: 2-aminobutyric acid; T: allothreonine. Small letters: D-amino acids; E.c.: Escherichia coli ATCC 25922, K.p.: Klebsiella pneumoniae ATCC 43816. MIC in μM can be found in the SI. bExperiment ran once.

of the amine and hydroxyl functions, Dab(βOH)3 was replaced by Dab (31), AlloThr (32), or Ser (33). In all cases, the better antibacterial activity observed for analogue 19 could not be reproduced and MIC equivalent or showing a 2fold difference with NOSO-95C were observed. Inhibition of the bacterial translation was equivalent to NOSO-95C for analogue 33 (Ser) and almost 2-fold lower for analogues 31 and 32 (Dab and AlloThr). Substitution by Gly (34) was also investigated to see if a greater decrease of the steric hindrance could improve the efficacy but a 2-fold decrease of MIC was observed on K. pneumoniae compare to NOSO-95C as well as a lower inhibition of the translation. Finally, a longer alkyl chain was tested by introducing Val (35) or Abu (aminobutyric acid, 36) but the same activities or a 2-fold improvement compare to NOSO-95C were observed and inhibition of the translation was reduced. Some amino acids (Gly, Pro, and Dhaa) have a strong influence on the shape of the peptide, and their replacement by Ala leads to the modification of the lateral chain but also of the structure of the peptide. When Gly4 (20) was replaced by Ala, a significant decrease of the inhibition of the translation and of the antibacterial activity was observed. This result could be explained by a lower flexibility of the peptidyl chain due to the substitution of the α carbon leading to an increase of the energy needed by the peptide to adopt the right structure to bind to the target and/or cross the bacterial membranes. Regarding the proline, the five-membered ring induces a constrain in the peptide chain and usually favors turn geometry.48 This has been confirmed on an analogue of NOSO-95C bound to the bacterial ribosome.14 When Pro6 (22) was substituted, an antibacterial activity close to NOSO-

the same than for NOSO-95C (23, 26) or slightly decreased (17, 24) and the same (24) or 2-fold lower (17, 23, and 26) on K. pneumoniae. Inhibition of the bacterial translation was close to the one of the parent compound (0.39−0.83 μM) except for Lys8 (24) where a 3-fold decrease was observed. The close activity between these analogues and NOSO-95C could be explained by these positions not interacting directly with the target. Again, this has been confirmed by a cocrystallization study for Lys1, His7, and Lys8.14 As the study was done on a shorten analogue, interaction of Lys10 could not be studied, but based on these results, it seems that this position is not key for binding of NOSO-95C to the ribosome. Interestingly, when Dbt11 (27) was removed a better inhibition of the translation than NOSO-95C (0.36 versus 0.63 μM) but a 2-fold decrease of antibacterial activity (MIC) were observed. The decrease in antibacterial efficacy despite a better inhibition of the translation could be explained by the crossing of the membranes being less efficient. Recently, Richter et al. have shown that one of the parameters that could play a role in the crossing of the membranes of Gram-negative bacteria is the presence of primary amines.49 The lowest antibacterial efficacy of 27 despite a better inhibition of the translation could thus be explained by the removal of the primary amine leading to a lower crossing of the membranes. When Dab(βOH)3 was substituted (19) a more potent analogue than parent compound was obtained with a 4-fold gain on E. coli and K. pneumoniae and an inhibition of the bacterial translation very close to NOSO-95C (0.62 versus 0.63 μM). The better antibacterial activity could be explained by a better crossing of the bacterial membranes. With the aim of understanding better this result and especially the influence 7818



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95C (2-fold decrease) was observed for E. coli while a 5-fold increase of IC50 was measured. Replacing the proline should lead to a more flexible peptide which could cross the bacterial membranes more easily, explaining the better activity on E. coli despite a lower inhibition of the translation. Surprisingly though, an 8-fold decrease of activity on K. pneumoniae was observed. Finally, when replacing Dha9 by Ala (25) a strong decrease of antibacterial activity and an increase of the IC50 value were observed. It was then decided to check separately the influence of lateral chain substitution by introducing dehydroaminobutyric acid (DhAbu, 37) and of the double bond by introducing L- or D-Arg (38 and 39). Analogue 37 (DhAbu) was found to be slightly less efficient to inhibit the translation and less active on E. coli and K. pneumoniae (2-fold decrease for both). Replacement of Dha by L- or D-Arg (38 and 39) led to a 2- to 8-fold decrease of MIC and to the inhibition of the translation proving the importance of the double bond for antibacterial activity. This first SAR study has shown the strong importance of lateral chains of position 2 and 5 and interestingly has shown that Dab(βOH) in position 3 can be replaced by readily available alanine. Our next effort was focused on identifying the minimum active sequence for this peptide. The first step was the iterative removal of amino acids from the C-terminal position. Removing Dbt11 (40) or both Dbt11 and Lys10 (41) led to structures with the same in vitro antibacterial efficacy than NOSO-95C on K. pneumoniae and a 2-fold lower activity on E. coli. Inhibition of the translation was slightly better than NOSO-95C as shown by the lower IC50 (0.36 and 0.43 μM versus 0.63 μM for NOSO-95C). The next step was to remove Dha9 (42). In that case, a dramatic decrease of both antibacterial activity and inhibition of the translation was observed confirming the strong importance of this moiety. As expected, removing one more amino acid (43) did not restore the antibacterial efficacy. The shortening of NOSO-95C from the N-terminal side was then investigated. Removing the first amino acid (Lys1, 44) led to a significant decrease of the antibacterial efficacy and of the inhibition of the translation. As expected, removal of both Lys1 and Dab(βOH)2 (45) led to the same result (Table 3). In conclusion shortening NOSO-95C from N- or C-terminal side has highlighted the key role of Dha9 and Lys1 for antibacterial activity and a shorter structure keeping good

antibacterial activities has been identified when removing Dbt11 and Lys10. Starting from these first two studies, combination of the best modifications was investigated. As such, a shorter analogue missing Lys10 and Dbt11 and bearing an alanine in place of Dab(βOH)3 was synthesized (NOSO-95179 46, Figure 3). Its

Figure 3. Structure of NOSO-95179 46.

antibacterial efficacy on wild-type K. pneumoniae ATCC 43816 and E. coli ATCC 25922 was measured and was found to be very close to that for NOSO-95C (Table 4, entries 1 and 12). As explained in the Introduction, we were particularly interested in efficacy against carbapenem resistant Enterobacteriaceae (CRE). In a previous publication,14 antibacterial effect against a panel of these strains bearing various classes of carbapenemases (KPC, NDM, VIM, or OXA) was measured for NOSO-95179. These antibacterial efficacies were then compared with NOSO-95C (Table 4). Both compounds were active against all strains including highly resistant ones with MIC of 4−8 μg/mL against K. pneumoniae and 4−16 μg/mL against E. coli for NOSO-95179 and 2−8 μg/mL against K. pneumoniae and 4−8 μg/mL against E. coli for NOSO-95C. MIC on a highly resistant strain of E. cloacae was also measured and was found to be on the same range than K. pneumoniae and E. coli for both compounds (Table 4, entry 16). The propensity of bacteria to develop resistance to NOSO95179 was assessed by determining the spontaneous frequency of resistance (FoR) to the compound with E. coli ATCC 25922 and K. pneumoniae ATCC 43816. Mutants of E. coli and K. pneumoniae resistant to 4× MIC of NOSO-95179 were isolated at frequencies of 3.5 × 10−9 and 4.6 × 10−9, respectively. Selectivity of the inhibition of the translation between eukaryotes and prokaryotes was also evaluated. As described previously,14 NOSO-95179 showed an IC50 on the mammalian system 300-fold higher than the one measured on the bacterial system (167.7 versus 0.55 μM). The same assay was run on NOSO-95C, and in that case only a 16-fold difference was measured between the two (10.3 versus 0.63 μM). NOSO95179 being more selective than NOSO-95C for bacterial versus mammalian inhibition, a better safety profile could be expected. It was thus selected for further evaluation. In vitro cytotoxicity against mammalian HepG2 (Human hepatocellular carcinoma cell) was measured and no toxicity was observed up to 256 μM (higher dose tested). Hemolysis was also evaluated at 100 μM for NOSO-95C and NOSO95179 and was found to be 0.43 and 0.36%, respectively. Finally, compound 46 was evaluated in a neutropenic mouse lung infection model using a K. pneumoniae ATCC 13883 (Figure 4). The study was run for 8 h postinfection with

Table 3. Removal of Amino Acids from C- or N-Terminal Positions of NOSO-95C 1a MIC (μg/mL) cmpd 1 40 41 42 43 44 45

peptide sequence NOSO-95C NOSO-95C NOSO-95C NOSO-95C NOSO-95C NOSO-95C NOSO-95C

(1−11) (1−10) (1−9) (1−8) (1−7) (2−11) (3−11)

E.c.

K.p.

IC50 (μM)

8 16 16 >64 >64 >64 >64

4 4 4 >64 >64 >64 >64

0.63 ± 0.05 0.36 ± 0.08 0.43 ± 0.10 10.01 ± 6.8 9.60 ± 6.71 >40 >40

a

Abbreviations are as follows: Cmpd: compound; MIC: minimum inhibitory concentration; E.c.: Escherichia coli ATCC 25922, K.p.: Klebsiella pneumoniae ATCC 43816. MIC in μM can be found in the SI. 7819



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Table 4. Antibacterial Activity of 46 and 1 and Comparators against Enterobacteriaceaea MIC (μg/mL) entry

strain

genotype

46

1

CTR

CIP

GEN

IPM

PMB

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

K. pneumoniae ATCC 43816 K. pneumoniae ATCC 13883 K. pneumoniae ATCC BAA-1905 K. pneumoniae ATCC BAA-1904 K. pneumoniae NCTC 13438 K. pneumoniae NCTC 13439 K. pneumoniae ATCC BAA-2146 K. pneumoniae ATCC BAA-2472 K. pneumoniae ATCC BAA-2473 K. pneumoniae NCTC 13443 K. pneumoniae NCTC 13442 E. coli ATCC 25922 E. coli ATCC BAA-2340 E. coli ATCC BAA-2452 E. coli ATCC BAA-2469 E. cloacae ATCC BAA-2468

WT WT KPC-2 KPC-3 KPC-3 VIM-1 NDM-1 NDM-1 NDM-1 NDM-1 OXA-48 WT KPC NDM-1 NDM-1 NDM-1

4 4 8 4 8 4 4 4 4 4 4 16 16 16 16 4

4 4 4 2 4 2 2 4 4 4 8 8 8 8 4 4

≤0.125 64 >64 >64 >64 >64 >64 >64 >64 8 64 >64 >64 >64

≤0.125 0.5 >64 0.5 >64 32 >64 >64 >64 >64 8 64 64 >64

0.25 1 32 16 2 1 >64 >64 >64 >64 0.25 0.5 1 >64 >64 >64

1 2 >64 64 >64 64 >64 >64 >64 >64 >64 0.5 32 >64 >64 >64

0.5 1 0.5 0.5 0.5 0.5 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

a Abbreviations are as follows: MIC: minimum inhibitory concentration; ceftriaxone (CTR), ciprofloxacin (CIP), gentamicin (GEN), imipenem (IPM), and polymyxin B (PMB).

noncommercial amino acid (Dab(βOH)3) can be replaced by readily available alanine while positions 2 and 5 are key for activity. After finding the minimum active sequence and combining best modifications, a potent analogue NOSO95179 46 has been identified showing promising in vitro antibacterial activities, in vivo efficacy, and no cytotoxicity. This new compound will be used as a starting point for a lead optimization campaign in order to improve even further its in vitro and in vivo antibacterial efficacy.

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EXPERIMENTAL SECTION

Chemistry. Materials. All commercially available reagents were used without further purification unless otherwise noted. Resins, HBTU, and protected amino acids were purchased from Chem Impex International Inc. (Wood dale, IL, U.S.A.). For Marfey’s analyses racemic mixtures, D and L enantiomers of Lys, Orn, Pro, His, as well as Gly and 1,4-diaminobutane were purchased from Bachem (Germany). The four diastereoisomers of Dab(β−OH) and the two diastereoisomers of the dipeptide Lys-(Z)-Dha were synthesized. All other reagents and solvents, including of HPLC grade, were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Purification of peptides was performed on a HPLC Agilent 1260 Infinity system with a Waters Symmetry semi preparative C18 column (7 μm, 7.8 mm × 300 mm). 50 μL of peptide solution (300 mg/mL) were injected, the flow rate was set up at 2.5 mL/min and UV detection was made at 230 nm. The following mobile phases were used: A: 0.1% trifluoroacetic acid in water; B: acetonitrile. The following gradient was used: 0 to 15% of B in A from 0 to 15 min. Analytical LC-MS of peptides was performed on the same system with a Waters Symmetry analytical C18 column (5 μm, 4.6 mm × 150 mm). A total of 10 μL was injected, the flow rate was set up at 0.7 mL/min, and UV detection was made at 230 nm. The following mobile phases were used: A: 0.2% heptafluorobutyric acid in water; B: acetonitrile. The following gradient was used: 20 to 50% of B in A from 0 to 15 min. ESI-LC-MS data were obtained in the positive mode on an Agilent 1260 Infinity system (Agilent 6120 Quadrupole LC/MS, 1260 Quaternary Pump, 1260 ALS, 1260 TCC, 1260 DAD VL, 1260 FC-AS). Analytical LC-MS for synthesis of protected Dab(βOH)6 was run using the same conditions, but UV detection was made at 230 nm. The following mobile phases were used: A: 0.1% trifluoroacetic acid in water; B: acetonitrile.

Figure 4. In vivo efficacy of 46 in lung infection model challenged with K. pneumoniae ATCC 13883. One-way ANOVA; Dunnett’s comparison versus vehicle; ns: not significant; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001.

ciprofloxacin as positive control (MIC = 0.5 μg/mL, 30 mg/kg injected intravenously). NOSO-95179 (MIC = 4 μg/mL) was injected at 20, 40, or 80 mg/kg subcutaneously. This resulted in a significant 2- and 3-log colony-forming unit (CFU) reduction per mL in bacterial burden in the lung compare to the vehicle when dosed once at 40 and 80 mg/kg, respectively, and a nice dose−response effect was observed. Ciprofloxacin also demonstrated a significant reduction. As presented before, the efficacy of compound 46 in a similar model using a CRE was very promising.14

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CONCLUSION The first total synthesis of NOSO-95C 1 has been achieved after developing the synthesis of two original building-blocks. A structure−activity relationships study has then been set up to evaluate the influence of lateral chains, structure, and size of the molecule. This study has shown that one of the 7820



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HRMS analyses were made by direct introduction in mass spectrometer Synapt G2-S (Waters, SN: UEB205) equipped with ESI. Mass spectra were recorded on positive or negative mode between 100 and 1500 Da. The capillary voltage was 2000 V, and the cone voltage was 30 V. The temperature of the source and of desolvation were respectively 120 and 250 °C. Data were analyzed using Masslynx 4.1. For NMR (nuclear magnetic resonance) spectra, chemical shifts are expressed in parts per million (ppm) with the solvent resonance as the internal standard (1H NMR: DMSO-d6: 2.50 ppm; D2O: 4.79 ppm; CDCl3: 7.26 ppm; 13C NMR: DMSO-d6: 39.5 ppm, CDCl3: 77.2 ppm). Spin multiplicity is described by the following abbreviations: s = singlet, d = doublet, t = triplet, q = quadruplet, qu = quintuplet, m = multiplet, dd = doublet of doublet, dt = doublet of triplet, and br = broad. Coupling constants are expressed in Hertz (Hz). For the synthesis of protected Dab(βOH) and NOSO-95C, 1H NMR spectra were recorded at 298 or 343 K on Brüker Avance III 600 MHz NMR spectrometer, using QXI Probe. 13C NMR spectra were recorded on the same instrument at 150 MHz. For the dipeptide Lys-Dha synthesis 1H NMR spectra were recorded at 298 K on a Brüker Ultra shield 400 plus (400.13 MHz) instrument, 13C NMR spectra were recorded on the same instrument at 100.6 MHz, and 31P NMR spectra were recorded on the same instrument at 161.99 MHz (the chemical shifts are reported from H3PO4 85% as external standard). For comparison of chemical shifts between natural and synthetic NOSO-95C NMR studies were done on a Brü ker Avance spectrometer operating at 700 MHz equipped with a cryoprobe.15 The sample was solubilized in water (95/5, H2O/D2O, v/v), data were recorded at 280 K, and 1H chemical shifts are expressed with respect to sodium 4,4-dimethyl-silapentane-1-sulfonate according to IUPAC recommendations. Marfey’s Analyses. Marfey’s analysis of NOSO-95C 1 was run as follows: a solution of Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA) in acetone (0.035 M) was prepared. This solution was added to a cleaved peptide’s solution or to a solution of deprotected amino acids in water (50 mM) followed by addition of a 1 N solution of sodium hydrogen carbonate in water (10.0 equiv). If formation of a solid was observed, DMSO was added until obtaining a clear solution. The mixture was heated at 40 °C for 1.5 h and then cooled to room temperature. The analytical sample was prepared with 40 μL of the solution, 40 μL of 2 M aqueous HCl, and 40 μL of DMSO. Analytical LC-MS of FDAA-derivatized samples was performed on a HPLC Agilent 1260 Infinity system with a Waters Symmetry analytical C18 (5 μm, 4.6 mm × 150 mm) and an Agilent Eclipse Plus C18 (3.5 μm, 4.6 mm × 100 mm) column. A total of 30 μL of the analytical sample was injected, the flow rate was set up at 1.0 mL/min, and UV detection was made at 340 nm. The following mobile phases were used: A: 0.1% trifluoroacetic acid in water; B: acetonitrile. The following gradient was used: 0 to 60% of B in A from 0 to 150 min. ESI-LC-MS data were obtained in the positive mode on an Agilent 1260 Infinity system (Agilent 6120 Quadrupole LC/MS, 1260 Quaternary Pump, 1260 ALS, 1260 TCC, 1260 DAD VL, 1260 FCAS). For Marfey’s analysis during the synthesis of protected Dab(βOH) 6, protected intermediates were first deprotected via heating a solution of the intermediate in aqueous 6 N HCl solution at 100 °C for 1 h. The solution was then lyophilized, and the solid obtained was dissolved in water (50 mM solution). Marfey’s analysis was then run as described above but using Nα-(2,4-dinitro-5-fluorophenyl)-Lvalinamide (FDVA). Analytical LC-MS of FDVA-derivatized samples was performed on the same conditions but with a Waters Symmetry analytical C18 (5 μm, 4.6 mm × 150 mm) column, injection of 10 μL of the analytical sample, flow rate set up at 0.7 mL/min, and a gradient of 0 to 55% of B in A from 0 to 110 min. General Procedure for Solid-Phase Peptide Synthesis (SPPS). All reactions were carried out in polypropylene empty reservoirs (15 mL) fitted with polyethylene frits and a polytetrafluoroethylene stopcock at room temperature. Loading of first amino acid: For 1 and analogues 17−26, 28−39, 44 and 45, 1,4-diaminobutane trityl resin (100 mg, 0.77 mmol/g) was

swelled in DCM (3.0 mL) for 15 min and rinsed for 20 s with DMF (2.5 mL). The Fmoc-protected amino acid (3.0 equiv), DMF (1.3 mL), and then DIPEA (4.0 equiv) were added to the resin, and the reservoir was shaken until full dissolution. HBTU (2.9 equiv) was added and the reservoir was shaken for 120 min. The reaction mixture was filtered, the coupling repeated once, and the resin was washed with DMF (3 × 2.5 mL, 0.5 min), MeOH (1 × 2.5 mL, 0.5 min), and DCM (6 × 2.5 mL, 0.5 min). For analogues 27, 40−43, and 46, 2chlorotrityl chloride resin (75 mg, 1.0 mmol/g) was swelled in DCM (3.0 mL) for 30 min. DCM was removed, and then a solution of Fmoc-protected amino acid (3.0 equiv) and DIPEA (4.0 equiv) in DCM (1.3 mL) was added. The reservoir was shaken for 240 min, the reaction mixture was filtered, the coupling was repeated once, and the resin was washed with DMF (3 × 2.5 mL, 0.5 min). A mixture of DCM/MeOH/DIPEA (80/15/5, 2.5 mL) was added to the resin, the reservoir was shaken for 10 min, the reaction mixture was filtered, the capping was repeated once, and the resin was washed with DMF (3 × 2.5 mL, 0.5 min), MeOH (1 × 2.5 mL, 0.5 min), and DCM (6 × 2.5 mL, 0.5 min). Fmoc removal was achieved with a solution of DMF/piperidine (80:20, 4.0 mL, 20 min × 2), and then the resin was washed with DMF (5 × 2.5 mL, 0.5 min), MeOH (1 × 2.5 mL, 0.5 min), DCM (1 × 2.5 mL), and DMF (2 × 2.5 mL, 0.5 min). Addition of next amino acids was achieved using the following conditions: Fmoc-protected amino acid (3.0 equiv), DMF (1.3 mL), and then DIPEA (4.0 equiv) were added to the resin, and the reservoir was shaken until full dissolution. HBTU (2.9 equiv) was added, and the reservoir was shaken for 120 min. The reaction mixture was filtered, the coupling was repeated once, and the resin was washed with DMF (3 × 2.5 mL, 0.5 min), MeOH (1 × 2.5 mL, 0.5 min), and DCM (6 × 2.5 mL, 0.5 min). Cleavage from the resin and global deprotection was achieved using the following conditions: after the last Fmoc removal the resin was rinsed carefully with DCM (5 × 2.5 mL, 2 min) and dried overnight (18 h) at room temperature. 2.0 mL of cleavage cocktail were freshly prepared (TFA/H2O/TIS, 85/7.5/7.5) and added to the resin. The mixture was shaken for 3 h, and the solution was added dropwise to tubes containing 30 mL of cold (0 °C) TBME. This step was repeated once, the cold mixture was stirred at 0 °C for 30 min, and the tubes were centrifuged (2,200g, 5 min). The supernatant was discarded, and the solid was washed with cold TBME (10 mL) and then centrifuged again (2,200g, 5 min). The crude product obtained was air-dried (3 h) at room temperature, dissolved in water, and freeze-dried. Crude peptides were purified on semi preparative HPLC. Pure fractions were combined and lyophilized, and the pure peptide was analyzed on analytical LC-MS and by HRMS (cf the Supporting Information). All peptides were of purity >95%. For the lung infection model, TFA salt of NOSO-95179 was exchanged for HCl salt. NOSO-95179 (TFA salt) was dissolved in aqueous HCl solution (0.05 M) at a concentration of 20 mg/mL. The solution obtained was freeze-dried, and this step was repeated twice. HCl salt of NOSO-95179 was obtained with a good purity (96.6%, LC-MS). TFA content was measured in the sample using 19F NMR and was found to be 90%). LC-MS (8 min gradient of 0 to 80% B in A): Rt = 2.30 min, [M + H]+ = 135; 1H NMR (D2O, 600 MHz, mixture of 2 diastereoisomers 7821



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for 18 h. The suspension obtained was filtered off to give 6 as a white powder (22.9 g, 73% yield). LC-MS (15 min gradient of 0 to 30% B in A): Rt = 19.66 min, 96% (254 nm), [M+H-Boc−CH(CH3)2]+ = 357; 1H NMR (DMSO-d6, 600 MHz, 343 K) δ 1.43 (s, 12H), 1.47 (s, 3H), 3.36−3.41 (m, 1H), 3.54−3.59 (m, 1H), 4.22−4.25 (m, 2H), 4.30−4.33 (m, 2H), 4.38 (br s, 1H), 7.30−7.34 (m, 2H), 7.39−7.44 (m, 2H), 7.58 (br s, 1H), 7.69−7.72 (m, 2H), 7.87 (d, J = 7.8, 2H); 13 C NMR (DMSO-d6, 150 MHz, 343 K):δ 27.8, 46.5, 46.8, 65.7, 72.8, 78.9, 92.9, 119.6, 124.8, 126.7, 127.2, 140.4, 143.5, 151.0, 155.5, 170.4. The regioselectivity of protections was determined using HMBC and HSQC analyses. A clear signal was observed in HMBC between the CHα (4.25 ppm) and the CO of the Fmoc protecting the amine in the α position (155.5 ppm) proving the regioselectivity of the protections; Marfey’s analysis: Rt = 96.19 min (2S,3S), 100% (340 nm), [M + H]+ = 695.15. (Z)-5-[[N,N′-bis(tert-Butoxycarbonyl)carbamimidoyl]amino]-2[[(2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9ylmethoxycarbonylamino)hexanoyl]amino]pent-2-enoic acid (7). A total of 1 L of 0.8 M CaCl2 solution in a mixture of iPrOH/H2O (7/3) was prepared (CaCl2 was dissolved first in H2O and then iPrOH was added and the mixture was stirred for 2 h at room temperature). This solution should be single phase and clear. (Z)-16 (30.0 g, 35.8 mmol, 1.0 equiv) was dissolved in the 0.8 M CaCl2 solution (580 mL). The mixture was stirred at room temperature for 20 min and then was cooled to 0 °C and aqueous NaOH (1 M, 71.6 mL, 71.6 mmol, 2.0 equiv) was added dropwise. The mixture was stirred at room temperature for 16 h, and then EtOAc and saturated NH4Cl aqueous solution were added. Aqueous phase was extracted twice with EtOAc. The combined organic phases were washed with brine. Organic phase was concentrated under vacuum without being dried over a drying agent. The crude mixture was purified by column chromatography. (Z)-7 was obtained as a white foam (17.0 g, 58% yield, >95% purity (LC-MS)). 1H NMR (DMSO-d6): δ 1.35−1.46 (m, 31H); 1.56−1.72 (m, 2H); 2.24 (brs, 2H); 2.90 (brs, 2H); 3.34− 3.39 (m, 2H); 4.09−4.13 (m, 1H); 4.18−4.30 (m, 3H); 6.26−6.35 (m, 1H); 6.74−6.79 (m, 1H); 7.28−7.39 (m, 2H); 7.40−7.43 (m, 2H); 7.57−7.60 (m, 1H); 7.69−7.75 (m, 2H); 7.83−7.89 (m, 2H); 8.32 (brs, 1H); 8.94 (brs, 1H); 11.49 (brs, 1H); 13C NMR (DMSOd6):δ 22.9, 27.5, 27.9, 28.2, 29.2, 31.6, 46.6, 54.9, 65.6, 77.3, 78.1, 82.8, 120.0, 121.3, 125.3, 127.0, 127.2, 127.6, 128.9, 140.7, 143.7, 143.8, 151.9, 155.2, 155.5, 156.0, 163.0, 170.2; LC-MS (Column: Kinetex (2.6 μm, 3 mm × 750 mm), gradient from 5 to 95% AcCN (0.1% TFA) in H2O (0.1% TFA) over 8 min.): Rt = 4.72 min, 100% [M + H]+ = 823. 2-(Benzyloxycarbonylamino)-2-hydroxyacetic acid (9). A solution of benzyl carbamate (90.0 g, 0.59 mol, 1.0 equiv) and monohydrate dihydroxyacetic acid 8 (72.1 g, 0.65 mol, 1.1 equiv) in toluene (840 mL) was introduced in a 2 L flask, and the solution was heated at 40 °C for 1.5 h. Half of the solvent was concentrated under reduce pressure. Toluene (540 mL) was added, and half of the solvent was concentrated under reduced pressure. Toluene (540 mL) was added, and the reaction was stirred at 40 °C for 2 h and then cooled to 20 °C. The white solid obtained was filtered, rinsed with toluene, and dried under vacuum. The expected compound 9 (133.0 g, quant. yield, 95% purity (1H NMR)) was obtained as a white solid. Analyses were in accordance with literature data.51 1H NMR (DMSO-d6): δ 5.03 (s, 2H); 5.20 (d, J = 9.0 Hz, 1H); 7.27−7.54 (m, 5H); 8.12 (d, J = 8.7 Hz, 1H); 13C NMR (DMSO-d6):δ 65.5, 73.2, 127.8, 128.3, 136.8, 155.4, 171.0. Methyl 2-(benzyloxycarbonylamino)-2-methoxy-acetate (10). 2(Benzyloxycarbonylamino)-2-hydroxyacetic acid 9 (133.0 g, 0.59 mol, 1.0 equiv) was diluted in methanol (480 mL). Trimethyl orthoformate (TMOF, 129.3 mL, 1.18 mol, 2.0 equiv) and HCl in methanol (1.25 M, 24 mL, 0.03 mol, 0.05 equiv) were added successively. The mixture was stirred at 56 °C for 40 min. Solvent was concentrated under reduce pressure, and Et2O (600 mL) was added. If necessary starting material was filtered off. The filtrate was concentrated and dried under reduce pressure. 10 was obtained as a white solid (149.6 g, crystallization can take long). Analyses were in accordance with literature data.51 1H NMR (DMSO-d6): δ 3.25 (s,

70:30): δ 3.22 (dd, J = 10.2 and 13.2 Hz, 0.3 H); 3.34−3.47 (m, 1.7H); 4.08 (d, J = 4.8 Hz, 0.3H); 4.24 (d, J = 3.0 Hz, 0.7H); 4.46 (td, J = 3.0 and 10.2 Hz, 0.7H); 4.48−4.52 (m, 0.3H); 13C NMR (D2O, 150 MHz, mixture of 2 diastereoisomers):δ 42.6; 43.4; 57.7; 66.9; 67.4; 170.4; Marfey’s analysis: 2S,3S: Rt = 96.01 min, 73%; 2R,3S: Rt = 97.84 min, 27%. ((2S,3S)-2-Amino-4-(tert-butoxycarbonylamino)-3-hydroxy-butanoic acid)2Cu (4). (2S,3S)-2,4-Diamino-3-hydroxy-butanoic acid (3) (53 g, ∼160 mmol) was put in a 2 L round-bottom flask and dissolved in water (250 mL). NaOH (3.0 equiv., 480 mmol, 19.0 g) was added portion wise (slightly exothermic). The mixture was stirred until dissolution of the solids, and then a solution of CuSO4·5H2O (0.5 equiv., 80 mmol, 20.0 g) in water (125 mL) was added slowly. The dark blue solution was heated at 110 °C for 30 min and then was cooled down to room temperature. A solution of Boc2O (2.0 equiv., 320 mmol, 69.7 g) in dioxane (275 mL) was added and the reaction stirred at room temperature for 3 days. A solution of Boc2O (0.5 equiv., 80 mmol, 17.4 g) in dioxane (60 mL) was added again, and the mixture was stirred at room temperature for 24 h. The suspension obtained was filtered. The pale blue solid obtained was rinsed with water (∼700 mL) and Et2O (∼300 mL) and then dried to give 4 as a pale blue solid (34.2 g, 40% yield over 2 steps). (2S,3S)-4-(tert-Butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-hydroxy-butanoic acid (5). ((2S,3S)-2Amino-4-(tert-butoxycarbonylamino)-3-hydroxy-butanoic acid)2Cu (4; 17.1 g, 32.0 mmol) was suspended in water (300 mL). A solution of Na2EDTA (1.5 equiv., 48.0 mmol, 17.8 g) and NaOH (3.0 equiv., 96.0 mmol, 3.8 g) in water (300 mL) was added. The mixture was stirred at room temperature for 4 h until full dissolution of the suspension. The solution obtained was cooled to 0 °C, and then a solution of FmocOSu (2.5 equiv., 80.0 mmol, 27.0 g) in dioxane (500 mL) was added slowly. At the end of the addition, Na2CO3 (2.5 equiv., 80.0 mmol, 8.5 g) was added, and the mixture was raised to room temperature and then stirred at room temperature for 18 h. The limpid blue solution obtained was washed with Et2O (4 × 200 mL) and then cooled in an ice bath. Aqueous 1 N HCl was added slowly until pH 3−4 (∼250 mL). This aqueous phase was extracted with AcOEt (5 × 200 mL). Organic phases were combined, washed with brine (2 × 150 mL), dried over MgSO4, filtered, and concentrated to give a pale yellow oil. AcCN (200 mL) was added and the mixture stirred at room temperature overnight. The suspension was filtered, and the solid rinsed with AcCN (100 mL) and then dried to give 5 as white powder (29.1 g, quant. yield). LC-MS (15 min gradient of 0 to 30% B in A): Rt = 13.96 min, 95% (254 nm), [M+H-Boc]+ = 357; 1H NMR (DMSO-d6, 600 MHz, 343 K): δ 1.39 (s, 9H); 3.00−3.04 (m, 1H); 3.12−3.20 (m, 1H); 3.85−3.88 (m, 1H); 4.00−4.13 (m, 1H); 4.22−4.25 (m, 1H); 4.28−4.31 (m, 2H); 6.61 (br s, 0.8H); 7.33 (t, J = 7.2 Hz, 2H); 7.42 (t, J = 7.2 Hz, 2H); 7.71 (d, J = 7.2 Hz, 2H); 7.87 (d, J = 7.2 Hz, 2H); 13C NMR (DMSO-d6, 150 MHz, 343 K):δ 27.9; 42.9; 46.5; 57.3; 65.7; 70.0; 77.6; 119.6; 124.9; 126.7; 127.2; 140.4; 143.5; 143.5; 151.3; 155.4; 155.6; 171.1; Marfey’s analysis: Rt = 95.60 min (2S,3S), 100% (340 nm), [M + H]+ = 695.15. (2S)-2-[(5S)-3-tert-Butoxycarbonyl-2,2-dimethyl-oxazolidin-5yl]-2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (6). (2S,3S)-4-(tert-Butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-hydroxy-butanoic acid (5; 29.1 g, 63.6 mmol) was suspended in a mixture of acetone and 2,2-dimethoxypropane (1:1, 480 mL). The suspension was cooled to 0 °C, and then BF3·OEt2 (catalytic, 900 μL) was added dropwise. The reaction was stirred for 2 h. An aqueous saturated solution of NaHCO3 (200 mL), AcOEt (400 mL), and water (300 mL) were added, and the phases were separated. The aqueous phase was extracted with AcOEt (2 × 200 mL). The organic phases were combined, washed with aqueous 0.1 N HCl (200 mL) and brine (200 mL), dried over MgSO4, filtered, and concentrated. The pale yellow oil obtained was dissolved in Et2O (100 mL), the solution was cooled to 0 °C, and then hexane (400 mL) was added. Formation of a suspension was observed while adding hexane. At the end of the addition, a sticky solid was observed at the bottom of the flask. Et2O was added at room temperature, and the mixture was triturated to obtain a white solid which was triturated 7822



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3H); 3.65 (s, 3H), 5.07 (s, 2H); 5.14 (d, J = 9.1 Hz, 1H); 7.30−7.38 (m, 5H); 8.45 (d, J = 8.7 Hz, 1H); 13C NMR (DMSO-d6): δ 52.1, 54.7, 65.9, 80.4, 127.8, 128.3, 128.5, 136.5, 155.8, 167.9. Methyl 2-(benzyloxycarbonylamino)-2-diethoxyphosphoryl-acetate (11). A 2 L three-necked round-bottom flask equipped with a dropping funnel and a condenser was dried by three heating +vacuum/argon cycles. 10 (149.5 g, 0.59 mol, 1.0 equiv) was introduced under argon followed by anhydrous toluene (720 mL). Three drops of concentrated sulfuric acid were added. Phosphorus trichloride (PCl3, 61.8 mL, 0.71 mol, 1.2 equiv) was introduced in the dropping funnel. The mixture was heated at 75 °C, and PCl3 was added over 1 h at this temperature. At the end of the addition, the mixture was stirred at 75 °C for 13 h. After cooling to room temperature, the solid was filtered off and the filtrate was concentrated under vacuum to remove excess PCl3. The crude mixture was diluted in anhydrous toluene (720 mL) under argon. Triethyl phosphite (111 mL, 0.65 mol, 1.1 equiv) was then added, and the mixture was stirred at 75 °C for 2 h and then at 90 °C for 30 min. The reaction mixture was cooled to room temperature, and solvent and excess triethyl phophite were removed under vacuum. The crude mixture was dissolved in EtOAc. Organic phase was washed twice with saturated Na2CO3, dried over MgSO4, filtered, and concentrated under vacuum. The crude mixture was precipitated in Et2O. The suspension was filtered off, and the solid was dried under vacuum to give 11 (165.1 g, 77% yield over 2 steps, 90% purity (1H NMR)) as a white solid. Analyses were in accordance with literature data.51 1H NMR (CDCl3): δ 1.27−1.38 (m, 6H); 3.79−3.90 (s, 3H); 4.10−4.25 (m, 4H); 4.90 (dd, J = 9.2 and 22.4 Hz, 1H); 5.09−5.20 (m, 2H); 5.60 (brs, 1H); 7.31−7.40 (m, 5H); 13C NMR (CDCl3):δ 16.3, 51.9, 53.2, 63.6, 67.4, 128.2, 128.3, 128.6, 128.8, 135.9, 155.6, 155.7, 167.4; 31P NMR (CDCl3):δ 15.85. Methyl 2-[[(2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9ylmethoxycarbonylamino)hexanoyl]amino]-2-diethoxyphosphoryl-acetate (12). Methyl 2-(benzyloxycarbonylamino)-2-diethoxyphosphoryl-acetate 11 (20.1 g, 55.7 mmol, 1.0 equiv) was dissolved in EtOH (600 mL). 10% palladium on charcoal (2.0 g, cat.) was added, and the reaction mixture was stirred under H2 atmosphere for 8−14 h. Deprotection was monitored by 31P NMR (few drops of the reaction mixture were mixed with DMSO-d6 and analyzed). After completion the mixture was filtered through Celite. The filtrate was concentrated under vacuum. The crude was dissolved in DCM (stored over amylene) and concentrated under reduced pressure. The operation was repeated three times to remove traces of EtOH. The free amine was used directly in the next step (product is unstable). The crude product was dissolved in DCM (60 mL, stabilized over amylene and filtered through alumina). Fmoc-Lys(Boc)−OH (26.0 g, 55.7 mmol, 1.0 equiv) was added followed by PyBOP (29.0 g, 55.7 mmol, 1.0 equiv). The reaction mixture was cooled to 0 °C before adding DIPEA dropwise (29.0 mL, 167.1 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature for 2 h. After completion, the reaction mixture was diluted with EtOAc. The organic phase was washed twice with a 5% KHSO4 aqueous solution, twice with a saturated aqueous solution of NaHCO3, and once with brine. Organic phases were dried over MgSO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography. After concentration under vacuum, foam was obtained. To remove traces of EtOAc, the crude mixture was dissolved in CHCl3 and concentrated under reduce pressure to give 12 as a white foam (33.4 g, 89% yield, 95% purity (1H NMR)). 1H NMR (DMSO-d6): δ 1.16−1.24 (m, 6H); 1.37 (s, 13H); 1.48−1.63 (m, 2H); 2.89 (brs, 2H); 3.70 (s, 3H); 4.01−4.09 (m, 4H); 4.19− 4.26 (m, 3H); 4.96−5.08 (m, 1H); 6.78 (brs, 1H); 7.30−7.35 (m, 2H); 7.38−7.44 (m, 2H); 7.51−7.55 (m, 1H); 7.70−7.75 (m, 2H); 7.86−7.92 (m, 2H); 8.72−8.80 (m, 1H); 13C NMR (DMSO-d6):δ 16.1, 22.8, 28.2, 29.2, 31.5, 31.7, 46.6, 49.4, 50.9, 52.7, 52.7, 54.2, 63.1, 63.1, 63.3, 63.3, 65.6, 77.3, 120.1, 125.3, 127.0, 127.6, 140.7, 143.7, 143.8, 155.5, 155.9, 167.1, 172.7; 31P NMR (DMSO-d6):δ 16.24 tert-Butyl-N-[(tert-butoxycarbonylamino)-(3hydroxypropylamino)methylene]carbamate (14). 1,3-bis(tert-Bu-

toxycarbonyl)-2-methyl-2-thiopseudourea 13 (50.0 g, 172.4 mmol, 1.0 equiv) was dissolved in DMF (400 mL). Aminopropan-3-ol (52.7 mL, 689.6 mmol, 4.0 equiv) was added dropwise followed by dimethylaminopyridine (2.1 g, 17.2 mmol, 0.1 equiv). The reaction mixture was stirred at room temperature for 4 h. (Caution! MeSH is generated during the reaction). The reaction mixture was dissolved in Et2O (4 L). The organic phase was washed with aqueous 0.1 M AcOH (800 mL), saturated aqueous NaHCO3 (800 mL), H2O (800 mL), and brine (800 mL). The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. 14 was obtained as a white solid (51.1 g, quant. yield, 95% purity (1H NMR)). Analyses were in accordance with literature data.50 1H NMR (CDCl3): δ 1.47 (s, 9H); 1.50 (s, 9H); 1.67−1.72 (m, 2H); 3.57−3.64 (m, 4H); 13C NMR (CDCl3):δ 28.1, 28.2, 32.4, 32.6, 32.7, 32.8, 36.5, 36.7, 57.7, 79.5, 83.4, 153.2, 157.2, 162.9. tert-Butyl-N-[(tert-butoxycarbonylamino)-(3-oxopropylamino)methylene]carbamate (15). Caution: the aldehyde is unstable and cannot be stored. 1.2 equiv of the aldehyde was prepared for the Horner−Wadsworth−Emmons (1.0 equiv of 12). In a 2 L flask, 14 (20.5 g, 64.7 mmol, 1.2 equiv) was dissolved in DCM (410 mL, stabilized over amylene). Pyridine (31.3 mL, 388.2 mmol, 7.2 equiv) was added followed by Dess−Martin periodinane (29.7 g, 70.1 mmol, 1.3 equiv). The reaction was stirred at room temperature for 3 h. A saturated aqueous solution of Na2CO3 (600 mL) and Et2O (300 mL) was added. The mixture was stirred at room temperature for 10 min. The suspension obtained was filtered through Celite. Et2O (1.1 L) was added, and the organic phase was washed with water (3 × 1 L). The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. 15 was obtained as yellow oil (22.1 g, quant.) and used directly in the next step without purification. Analyses were in accordance with literature data.51 1H NMR (CDCl3): δ 1.45−1.54 (m, 18H); 2.73−2.82 (m, 2H); 3.70−3.75 (m, 2H); 9.82 (s, 1H). Methyl (Z)-5-[[N,N′-bis(tert-butoxycarbonyl)carbamimidoyl]amino]-2-[[(2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9ylmethoxycarbonylamino)hexanoyl]amino]pent-2-enoate (16). In a 500 mL flask under argon 12 (36.1 g, 53.4 mmol, 1.0 equiv) was dissolved in anhydrous AcCN (300 mL). Dry lithium chloride (2.7 g, 64.1 mmol, 1.2 equiv) was added, and the reaction mixture was stirred at room temperature for 30 min. Aldehyde 15 (22.1 g) was dissolved in anhydrous AcCN (40 mL). The solution was added to the reaction mixture, and then DIPEA (11.1 mL, 64.1 mmol, 1.2 equiv) was added dropwise. The reaction mixture was stirred at room temperature for 3−4 days. The reaction was monitored by 31P NMR (few drops of the reaction mixture were mixed with DMSO-d6 and analyzed). After completion, EtOAc (1 L) was added, and the organic phase was washed with H2O (100 mL). The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. After HPLC analysis, the Z/E ratio was determined as 86/14. The crude product was purified by column chromatography. (Z)-16 was obtained as white foam (30.0 g, 67% yield, 90% purity (1H NMR)). 1H NMR (DMSO-d6): δ 1.21−1.47 (m, 31H); 1.52−1.69 (m, 2H); 2.31−2.36 (m, 2H); 2.90 (brs, 2H); 3.32−3.39 (m, 2H); 3.64 (s, 3H); 4.07− 4.12 (m, 1H); 4.20−4.28 (m, 3H); 6.40−6.44 (m, 1H); 6.78 (brs, 1H); 7.30−7.34 (m, 2H); 7.39−7.42 (m, 2H); 7.53 (d, J = 8.0 Hz, 1H); 7.70−7.73 (m, 2H); 7.85−7.90 (m, 2H); 8.37−8.41 (m, 1H); 9.32 (s, 1H); 11.47 (s, 1H); 13C NMR (DMSO-d6):δ 22.7, 27.3, 27.5, 27.9, 28.2, 29.2, 31.5, 46.6, 51.9, 54.4, 65.6, 77.3, 78.1, 82.8, 120.1, 125.3, 127.0, 127.6, 128.0, 132.9, 140.7, 143.7, 143.8, 151.8, 155.3, 155.5, 155.9, 163.0, 164.4, 171.3. Analyses of Synthetic Peptides. NOSO-95C 1. LC-MS: Rt = 13.51 min, [M + H]+ = 1264.6, purity: 96.5% (230 nm). For Marfey’s analysis a cleaved peptide solution was obtained as follow: a 1 μM solution of NOSO-95C in 6 N aqueous HCl was heated at 110 °C for 24 h. The solution was lyophilized and the solid obtained was dissolved in water to 50 mM. Procedure for Marfey’s analysis was then applied. In all cases, only one enantiomer was observed. All chiral centers were found to be of S configuration, except the chiral center of Orn which was found to be of R configuration (see the Supporting Information). 7823



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NOSO-95179 46 (TFA Salt). LC-MS: Rt = 12.26 min, [M + H]+ = 1021.5, purity: 97.78% (230 nm). NOSO-95179 46 (HCl Salt). LC-MS: Rt = 12.24 min, [M + H]+ = 1021.5, purity: 96.61% (230 nm). Biological Tests. Bacterial Strains and Antimicrobial Agents. Reference strains used in this study come from the American type culture collection (ATCC) and from the national collection of types cultures (NCTC). Ceftriaxone (Sigma-Aldrich, ref: 89434), ciprofloxacin (Sigma-Aldrich, ref: 1134335), gentamicin (Sigma-Aldrich, ref: G1397), imipenem (Sigma-aldrich, ref: IO160), and polymyxin B (Sigma-Aldrich, ref: 92283) were provided by the manufacturers as standard powders for ciprofloxacin and imipenem and as solution at 50 and 20 mg/mL for gentamicin and polymyxin B, respectively. Minimum Inhibitory Concentration (MIC). MIC values were determined using Clinical and Laboratory Standards Institute (CLSI) broth microdilution methodology and direct colony suspension as described in CLSI document M07-A10.46 Determination of Mutation Frequency. Bacterial strains were grown in antibiotic-free Luria−Bertani broth at 35 °C for 18 h. Approximately 109 CFU of each strain were plated in duplicate onto Mueller−Hinton agar plates containing NOSO-95179 concentration at 4× the MIC values. The plates were read after 48 h of incubation at 35 °C. The frequency of selected resistant mutants was calculated as the ratio of the number of bacteria growing divided by the number of bacteria in the original inoculum, which was calculated by plating several dilutions of the original inoculum. Bacterial Cell-Free Transcription-Translation Assays. The effect of Odilorhabdin’s analogues on in vitro bacterial protein synthesis was tested in the Expressway Cell-Free E. coli Expression System (Invitrogen). The gf p gene was amplified from the pCmGFP plasmid51 and cloned into pEXP5-CT TOPO vector (Thermo Fisher) and the resulting plasmid was used as a template for in vitro transcription-translation. The reactions were assembled and miniaturized following the manufacturer’s protocol and carried out in 50 μL in the wells of polystyrene black 96 half-well microplate (Greiner ref 675077) including the addition of feed buffer at 30 min of incubation to support optimal protein synthesis. Reactions were initiated by adding 1 μg of plasmid DNA, plates were incubated at 30 °C, and fluorescence was measured every 20 min (λex = 475 nm, λem = 520 nm) with a microplate reader. IC50 values were calculated at 1 h after addition of feed buffer using GraphPad Prism 6 Software (model: log(inhibitor) vs response − variable slope (four parameters)). Experiments were performed in duplicates. Cytotoxicity. HepG2 cell line derived from a hepatocellular carcinoma was obtained from the American Type Culture Collection (Rockville, MD, U.S.A.) and was cultured according to the supplier’s instructions. HepG2 cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) and 1% glutamine. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cell growth inhibition was determined by an MTS assay according to the manufacturer’s instructions (Promega, Madison, WI, U.S.A.). Briefly, the cells were seeded in 96-well plates (5 × 103 cells/well) containing 200 μL of growth medium. After 24 h of culture, the cells were treated with NOSO-95179 at 32, 64, 128, and 256 μg/mL. After 72 h of incubation, 40 μL of resazurin were added for 2 h before recording absorbance at 490 nm with a spectrophotometric plate reader. The percent cytotoxicity index [(OD490 treated/OD490 control) × 100] was calculated from three experiments. IC50 value corresponds to the concentration of compound that induced a decrease of 50% in absorbance of drug-treated cells compared with untreated cells. Experiments were performed in triplicate. Hemolytic Activity Assay. Mouse red blood cells were washed with 0.9% sodium chloride solution (saline solution) until the supernatant was clear after centrifugation and resuspended in saline solution to 10% (v/v). A total of 300 mL of the suspension was added to an equal volume of NOSO-95179 to give final concentrations of 256 mg/mL. Saline solution and ultrapure water were used as 0% and 100% hemolytic control, respectively. Microtubes were incubated at 35 °C for 45 min. Then, the microtubes were centrifuged, and the

supernatants were transferred to monitor the release of hemoglobin at 540 nm. Experiments were performed in triplicate. Mouse Lung Infection Model. Efficacy of NOSO-95179 (HCl salt) was assessed by VibioSphen (Labège, FR). All procedures were performed in accordance with the directive 2010/63/UE recommendations and with French veterinary authorities’ agreement. The in vivo design and procedures were approved by ethical committee (CEEA-122 2014-53). NOSO-95179 was tested against K. pneumoniae ATCC 13883 in a neutropenic mouse pulmonary infection model. Mice were allowed to acclimatize for 5 days and then rendered neutropenic by intraperitoneal injection of cyclophosphamide (150 mg/kg on day 4 and 100 mg/kg on day 1 before infection). Mice were infected by intranasal route (2 × 106 CFU/mouse). At 1 h post infection, mice received treatment with NOSO-95179 at 20, 40, or 80 mg/kg administered by subcutaneous (SC) route in a single dose in a volume of 10 mL/kg (6 mice per dose) or ciprofloxacin by intravenous (IV) route at 30 mg/kg in a volume of 10 mL/kg to serve as positive control. At 1 h post infection, one infected group was ethically euthanized, and the lungs were processed for pretreatment quantitative culture to determine Klebsiella burdens. At 8 h post infection, all remaining mice were ethically euthanized. Lungs were aseptically removed, homogenized, serially diluted, and plated on TSA (Tryptic Soy Agar) for CFU titers.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00790.

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NMR spectra of compounds 3−7 and 9−16, NOESY of Z and E Lys-Dha 7, analyses of natural and synthetic NOSO-95C 1 and synthetic NOSO-95179 46, HRMS of new compounds, MIC in μM, and inhibition curves for IVTT (PDF) Table with molecular formula strings associated with biological data (CSV)

AUTHOR INFORMATION

Corresponding Author

*Phone: 00334 11 71 61 82. E-mail: e.racine@nosopharm. com. ORCID

Renata Marcia de Figueiredo: 0000-0001-5336-6071 Jean-Marc Campagne: 0000-0002-4943-047X Emilie Racine: 0000-0002-4265-9669 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): M. G. and P. V.-G. are founders and shareholders of Nosopharm.

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ACKNOWLEDGMENTS This work has received financial support from OSEO and Region Languedoc-Roussillon under Grant Agreement No. A1010014J, and from DGA under Grant Agreement No. 122906117. The authors would like to thank André Aumelas for NMR analyses of peptides and Z/E determination of dipeptide Lys-Dha. 7824



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Figueiredo, R.; Midrier, C.; Gaudriault, S.; Givaudan, A.; Lanois, A.; Forst, S.; Aumelas, A.; Cotteaux-Lautard, C.; Bolla, J.-M.; Vingsbo Lundberg, C.; Huseby, D. L.; Hughes, D.; Villain-Guillot, P.; Mankin, A. S.; Polikanov, Y. S.; Gualtieri, M. Odilorhabdins, antibacterial agents that cause miscoding by binding at a new ribosomal site. Mol. Cell 2018, 70, 83−94. (15) Gualtieri, M.; Villain-Guillot, P.; Givaudan, A.; Pages, S. Novel Peptide Derivatives as Antibiotics. PCT Int. Appl., WO 2013045600A1, 2012. (16) Blanchard, S. C. A much-needed boost for the dwindling antibiotic pipeline. Mol. Cell 2018, 70, 3−5. (17) Merrifield, R. B. Solide Phase Peptide Synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149−2154. (18) Chan, W. C.; White, P. D. Basic procedures In Fmoc Solid Phase Peptide Synthesis, a Practical Approach, 1st ed.; Chan, W. C., White, P. D., Eds.; Oxford University Press: Oxford, U.K., 2000; pp 41−76. (19) Stepan, A. F.; Nguyen, T.-T.; Anderson, D.; Liang, H.; Zhanshan, Q.; Magee, T. V. Stereoselective synthesis of orthogonally protected β-hydroxy-α,χ-diamino butyric acids. Synlett 2011, 2011, 2499−2504. (20) Shaw, K. J.; Luly, J. R.; Rapoport, H. Routes to mitomycins. Chirospecific synthesis of aziridinomitosenes. J. Org. Chem. 1985, 50, 4515−4523. (21) Vassilev, V. P.; Uchiyama, T.; Kajimoto, T.; Wong, C.-H. Lthreonine aldolase in organic synthesis: Preparation of novel βhydroxy-α-amino acids. Tetrahedron Lett. 1995, 36, 4081−4084. (22) Gutierrez, M. L.; Garrabou, X.; Agosta, E.; Servi, S.; Parella, T.; Joglar, J.; Clapés, P. Serine hydroxymethyl transferase from Streptococcus thermophilus and L-threonine aldolase from Escherichia coli as stereocomplementary biocatalysts for the synthesis of βhydroxy-α,ω-diamino acid derivatives. Chem. - Eur. J. 2008, 14, 4647− 4656. (23) Sicher, J.; Rajsner, M.; Rudinger, J.; Eckstein, M.; Sorm, F. Amino-acids and peptides. XXVIII. Synthesis of threo- and erythroDL-α,γ-diamino-β-hydroxybutyric acid (γ-aminothreonine and γaminoallothreonine). Collect. Czech. Chem. Commun. 1959, 24, 3719−3729. (24) Inbar, L.; Lapidot, A. The structure and biosynthesis of new tetrahydropyrimidine derivatives in actinomycin D producer Streptomyces parvulus. Use of 13C- and 15N-labeled L-glutamate and 13 C and 15N NMR spectroscopy. J. Biol. Chem. 1988, 263, 16014− 16022. (25) Racine, E. Process for Preparing 2,4-Diamino-3-Hydroxy Butyric Acid Derivatives. PCT Int. Appl., WO 2015128504A1, 2015. (26) Bhushan, R.; Brückner, H. Marfey’s reagent for chiral amino acid analysis: a review. Amino Acids 2004, 27, 231−247. (27) Spetzler, J. C.; Hoeg-Jensen, T. Masked side-chain aldehyde amino acids for solid-phase synthesis and ligation. Tetrahedron Lett. 2002, 43, 2303−2306. (28) Kurtz, A. C. A simple synthesis of DL-citrulline. J. Biol. Chem. 1938, 122, 477−484. (29) Wiejak, S.; Masiukiewicz, E.; Rzeszotarska, B. A large scale synthesis of mono- and di-urethane derivatives of lysine. Chem. Pharm. Bull. 1999, 47, 1489−1490. (30) Isidro-Llobet, A.; Alvarez, M.; Albericio, F. Amino acidprotecting groups. Chem. Rev. 2009, 109, 2455−2504. (31) Isolation of the 2S,3R diastereoisomer was investigated by sending mother liquors directly to the next step. This trial was unsuccessful and unwanted 2S,3R diastereoisomer could not be isolated. (32) Breitholle, E. G.; Stammer, C. H. Synthesis of some dehydrophenylalanine peptides. J. Org. Chem. 1976, 41, 1344−1349. (33) Botes, D. P.; Viljoen, C. C.; Kruger, H.; Wessels, P. L.; Williams, D. H. Configuration assignments of the amino acid residues and the presence of N-methyldehydroalanine in toxins from the bluegreen alga, Microcystis aeruginosa. Toxicon 1982, 20, 1037−1042. (34) Stammer, C. H. Chemistry and Biology of Amino Acids, Peptides and Proteins; John Wright and Sons Ltd.: London, 1982; Vol. 6, pp 33−74.

ABBREVIATIONS USED Boc, tert-butyloxy carbonyl; Boc2O, di-tert-butyl dicarbonate; cat., catalytic; Cbz, carboxybenzyl; DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMEM, Dulbecco’s Modified Eagle Medium; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EDTA, ethylene diamine tetraacetic acid; Fmoc, fluorenylmethyloxycarbonyl; Fmoc-OSu, N-(9-fluorenylmethoxycarbonyloxy)succinimide; HBTU, (N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NOESY, nuclear Overhauser effect spectroscopy; ROESY, rotating-frame Overhauser spectroscopy; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; rt, room temperature; TFA, trifluoroacetic acid; TIS, triisopropyl silane.

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