Stereoselective Synthesis of meso- and l,l-Diaminopimelic Acids from Enone-Derived α-Amino Acids.

The stereoselective synthesis of meso-diaminopimelic acid (meso-DAP), the key cross-linking amino acid of the peptidoglycan cell wall layer in Gram-negative bacteria, and its biological precursor, l,l-DAP, is described. The key step involved stereoselective reduction of a common enone-derived amino acid by substrate- or reagent-based control. Overman rearrangement of the resulting allylic alcohols, concurrent alkene hydrogenation and trichloroacetamide reduction, and subsequent ruthenium-catalyzed arene oxidation completed the synthesis of each stereoisomer. The synthetic utility of this approach was demonstrated with the efficient preparation of an l,l-DAP-derived dipeptide.

Synthesis of Conformationally Constrained d-Glu-meso-DAP Analogs as Innate Immune Agonists.

The dipeptide d-Glu-meso-DAP (iE-DAP) is the minimal structural fragment capable of activating the innate immune receptor nucleotide-binding oligomerization domain protein (NOD1). The meso-diaminopimelic acid (meso-DAP) moiety is known to be very stringent in terms of the allowed structural modifications which still retain the NOD1 activity. The aim of our study was to further explore the chemical space around the meso-DAP portion and provide a deeper understanding of the structural features required for NOD1 agonism. In order to achieve the rigidization of the terminal amine functionality of meso-DAP, isoxazoline and pyridine heterocycles were introduced into its side-chain. Further, we incorporated the obtained meso-DAP mimetics into the structure of iE-DAP. Collectively, nine innovative iE-DAP derivatives additionally equipped with lauroyl or didodecyl moieties at the α-amino group of d-Glu have been prepared and examined for their NOD1 activating capacity. Overall, the results obtained indicate that constraining the terminal amino group of meso-DAP abrogates the compounds' ability to activate NOD1, since only compound 6b retained noteworthy NOD1 agonistic activity, and underpin the stringent nature of this amino acid with regard to the allowed structural modifications.

Facile Synthesis and Metabolic Incorporation of m-DAP Bioisosteres Into Cell Walls of Live Bacteria.

Bacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes cross-linked by cell wall transpeptidases (TP). While synthetic PG fragments containing l-lysine in the third position on the stem peptide are easier to access, those with meso-diaminopimelic acid (m-DAP) pose a severe synthetic challenge. Herein, we describe a solid phase synthetic scheme based on widely available building blocks to assemble meso-cystine (m-CYT), which mimics key structural features of m-DAP. To demonstrate proper mimicry of m-DAP, cell wall probes were synthesized with m-CYT in place of m-DAP and evaluated for their metabolic processing in live bacterial cells. We found that m-CYT-based cell wall probes were properly processed by TPs in various bacterial species that endogenously contain m-DAP in their PG. Additionally, we have used hybrid quantum mechanical/molecular mechanical (QM/MM) and molecular dynamics (MD) simulations to explore the influence of m-DAP analogs on the PG cross-linking. The results showed that the cross-linking mechanism of transpeptidases occurred through a concerted process. We anticipate that this strategy, which is based on the use of inexpensive and commercially available building blocks, can be widely adopted to provide greater accessibility of PG mimics for m-DAP containing organisms.

Synthesis of a meso-Oxa-Diaminopimelic Acid Containing Peptidoglycan Pentapeptide and Coupling to the GlcNAc-anhydro-MurNAc Disaccharide.

The syntheses of peptidoglycan (PG)-derived peptides containing meso-diaminopimelic acid (meso-Dap) are typically quite lengthy due to the need to prepare orthogonally protected meso-Dap. In this work, the preparation of the PG pentapeptide containing the isosteric analog meso-oxa-Dap is described. The synthesis relies on the ring opening of a peptide embedded aziridine via the attack of a serine residue. The pentapeptide was attached to a GlcNAc-anhydro-MurNAc disaccharide, to produce a putative substrate for the AmpG pore protein.

Practical spectrophotometric assay for the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase, a potential antibiotic target.

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] µM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] µM, phenylboronic acid (IC50 = 316 [± 23.6] µM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] µM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics.

Diaminopimelic acid (DAP) analogs bearing isoxazoline moiety as selective inhibitors against meso-diaminopimelate dehydrogenase (m-Ddh) from Porphyromonas gingivalis.

Two diastereomeric analogs (1 and 2) of diaminopimelic acid (DAP) bearing an isoxazoline moiety were synthesized and evaluated for their inhibitory activities against meso-diaminopimelate dehydrogenase (m-Ddh) from the periodontal pathogen, Porphyromonas gingivalis. Compound 2 showed promising inhibitory activity against m-Ddh with an IC50 value of 14.9µM at pH 7.8. The two compounds were further tested for their antibacterial activities against a panel of periodontal pathogens, and compound 2 was shown to be selectively potent to P. gingivalis strains W83 and ATCC 33277 with minimum inhibitory concentration (MIC) values of 773µM and 1.875mM, respectively. Molecular modeling studies revealed that the inversion of chirality at the C-5 position of these compounds was the primary reason for their different biological profiles. Based on these preliminary results, we believe that compound 2 has properties consistent with it being a lead compound for developing novel pathogen selective antibiotics to treat periodontal diseases.

Synthesis of conformationally constrained γ-D-glutamyl-meso-diaminopimelic acid derivatives as ligands of nucleotide-binding oligomerization domain protein 1 (Nod1).

Nod1, an important member of the pattern recognition receptor family, remains a virtually unexploited target. Harnessing its innate immune stimulatory properties still remains an unfulfilled goal of medicinal chemistry. Nucleotide-binding oligomerization domain protein 1 (Nod1) agonists have been shown to boost the inflammatory responses against pathogenic microbes and could thus constitute a new class of broad spectrum antimicrobial agents. To gain additional insight into the structure/activity relationships of Nod1 agonistic compounds, a series of novel, conformationally constrained γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) analogs have been designed and synthesized. Ramos-Blue cells expressing Nod1 were used to screen and validate our compounds for their Nod1-agonist activity. Their immunomodulatory properties were subsequently determined in vitro, by evaluating their capacity to induce pro-inflammatory cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC), by themselves and in synergy with lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand. The synthesized iE-DAP analogs were shown to possess immuno-enhancing properties as a result of their potent and specific Nod1-agonistic effect. The activity of the compound exhibiting the greatest capacity to induce pro-inflammatory cytokine release from PBMC surpassed that of lauroyl-γ-D-glutamyl-meso-diaminopimelic acid (C12-iE-DAP).

A facile synthesis of fully protected meso-diaminopimelic acid (DAP) and its application to the preparation of lipophilic N-acyl iE-DAP.

Synthesis of beneficial protected meso-DAP 9 by cross metathesis of the Garner aldehyde-derived vinyl glycine 1b with protected allyl glycine 2 in the presence of Grubbs second-generation catalyst was performed. Preparation of lipophilic N-acyl iE-DAP as potent agonists of NOD 1-mediated immune response from 9 is described.

(Acyloxy)alkoxy moiety as amino acids protecting group for the synthesis of (R,R)-2,7 diaminosuberic acid via RCM.

A new synthetic pathway is described to prepare asymmetrically protected 2,7-diaminosuberic acid. This strategy exploits (acyloxy)alkoxy promoiety as protecting group and RCM reaction using second generation Grubbs catalyst and provides the trans isomer of (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-(tert-butoxycarbonylamino)-8- methoxy-8-oxooct-4-enoic acid, which was in turn reduced to obtain (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino)- 2-(tert-butoxycarbonylamino)-8-methoxy-8-oxooctanoic acid.

Structure-activity relationships in nucleotide oligomerization domain 1 (Nod1) agonistic γ-glutamyldiaminopimelic acid derivatives.

N-acyl-γ-glutamyldiaminopimelic acid is a prototype ligand for Nod1. We report a detailed SAR of C(12)-γ-D-Glu-DAP. Analogues with glutaric or γ-aminobutyric acid replacing the glutamic acid show greatly attenuated Nod1-agonistic activity. Substitution of the meso-diaminopimelic (DAP) acid component with monoaminopimelic acid, L- or D-lysine, or cadaverine also results in reduced activity. The free amine on DAP is crucial. However, the N-acyl group on the D-glutamyl residue can be substituted with N-alkyl groups with full preservation of activity. The free carboxylates on the DAP and Glu components can also be esterified, resulting in more lipophilic but active analogues. Transcriptomal profiling showed a dominant up-regulation of IL-19, IL-20, IL-22, and IL-24, which may explain the pronounced Th2-polarizing activity of these compounds and also implicate cell signaling mediated by TREM-1. These results may explain the hitherto unknown mechanism of synergy between Nod1 and TLR agonists and are likely to be useful in designing vaccine adjuvants.

A simple chromatographic route for the isolation of meso diaminopimelic acid.

Meso diaminopimelic acid is an important noncoded amino acid found in Gram-negative bacterial peptidoglycan. In spite of its importance, this stereoisomer is not available commercially. A simple, economical procedure was developed for the isolation of pure meso diaminopimelic acid via an high-performance liquid chromatography separation. In our new approach, the underivatized three isomers of diaminopimelic acid were separated on a crown ether-based chiral stationary phase. For the structure identification, (1)H NMR spectroscopy was applied.

Synthesis of N-succinyl-L,L-diaminopimelic acid mimetics via selective protection.

The search for potential inhibitors that target so far unexplored bacterial enzyme mono-N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) has stimulated a development of methodology for quick and efficient preparation of mono-N-acylated 2,6-diaminopimelic acid (DAP) derivatives bearing the different carboxyl groups or lipophilic moieties on their amino group.

The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues.

The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 A of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical.

Synthesis of diaminopimelic acid containing peptidoglycan fragments and tracheal cytotoxin (TCT) and investigation of their biological functions.

Bacterial cell wall peptidoglycan (PGN) is a potent immunostimulator and immune adjuvant. The PGN of Gram-negative bacteria and some Gram-positive bacteria contain meso-diaminopimelic acid (meso-DAP), and we have recently shown that the intracellular protein Nod1 is a PGN receptor and recognizes DAP-containing peptides. In this study, we achieved the synthesis of DAP-containing PGN fragments, including the first chemical synthesis of tracheal cytotoxin (TCT), GlcNAc-(beta1-4)-(anhydro)MurNAc-L-Ala-gamma-D-Glu-meso-DAP-D-Ala, and a repeating-unit of DAP-type PGN, GlcNAc-(beta1-4)-MurNAc-L-Ala-gamma-D-Glu-meso-DAP-D-Ala. For the synthesis of PGN fragments, we first established a new synthetic method for an orthogonally protected meso-DAP derivative, and then we constructed the glycopeptide structures. The ability of these fragments to stimulate human Nod1, as well as differences in Nod1 recognition of the variety of synthesized ligand structures were examined. The results showed that the substitution of the N terminus of iE-DAP is necessary for stronger Nod1 recognition, but the structure of the substituent seems not to be strictly recognized. The importance of the carboxyl group at the 2-position of DAP for human Nod1 stimulation was also shown.

Stereoselective syntheses of 4-oxa diaminopimelic acid and its protected derivatives via aziridine ring opening.

Regio- and stereoselective aziridine ring opening with oxygen nucleophiles derived from serine and threonine provides a route to stereochemically pure 4-oxa-2,6-diaminopimelic acid (oxa-DAP) and its methyl-substituted derivatives. Oxa-DAP is a substrate of DAP epimerase, a key enzyme for biosynthesis of l-lysine and formation of peptidoglycan precursors. Orthogonally protected analogues of lanthionine and beta-methyllanthionine wherein oxygen replaces sulfur were prepared that could be used for solid-supported peptide synthesis to make oxa derivatives of lantibiotics.

A straightforward stereoselective synthesis of meso-, (S,S)- and (R,R)-2,6-diaminopimelic acids from cis-1,4-diacetoxycyclohept-2-ene.

A straightforward synthesis of meso-2,6-diaminopimelic acid (DAP) meso-1 was developed from 1,4-diacetoxycyclohept-2-ene (2) via an oxidative ring cleavage. Subsequently, an enantio-divergent synthesis of (S,S)- and (R,R)-1 was performed using a homochiral monoacetate 7 available from 2 by enzymatic desymmetrization.

Meso-diaminopimelic acid and meso-lanthionine, amino acids specific to bacterial peptidoglycans, activate human epithelial cells through NOD1.

Peptidoglycans (PGNs) are ubiquitous constituents of bacterial cell walls and exhibit various immunobiological activities. Two types of minimum essential PGN structures for immunobiological activities were chemically synthesized and designated as muramyldipeptide; N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), which are common constituents of both Gram-positive and Gram-negative bacteria, as well as most Gram-negative and some Gram-positive bacteria, respectively. Recently, intracellular receptors for MDP and iE-DAP have been demonstrated to be nucleotide-binding oligomerization domain (NOD)1 and NOD2, respectively. In this study, we demonstrated that chemically synthesized meso-DAP itself activated human epithelial cells from various tissues, through NOD1 to generate antibacterial factors, PGN recognition proteins and beta-defensin 2, and cytokines in specified cases, although the activities of meso-DAP were generally weaker than those of known NOD agonists. However, stereoisomers of meso-DAP, LL-DAP, and DD-DAP were only slightly activated or remained inactive, respectively. Synthetic meso-lanthionine, which is another diamino-type amino acid specific to PGN of the specified Gram-negative bacteria, was also recognized by NOD1. In human monocytic cells, in the presence of cytochalasin D meso-DAP induced slightly but significantly increased production of cytokines, although the cells did not respond to meso-DAP in the absent of cytochalasin D. Our findings suggest that NOD1 is a special sentinel molecule, especially in the epithelial barrier, allowing the intracellular detection of bacteria through recognizing meso-DAP or comparable moiety of PGN from specified bacteria in cooperation with NOD2, thereby playing a key role in innate immunity.

An efficient RCM-based synthesis of orthogonally protected meso-DAP and FK565.

A condensation--ring-close--ring-open sequence was employed for the synthesis of orthogonally protected meso-2,6-diaminopimelic acid, starting from easily accessible chiral synthons. Condensation of suitably protected L-allylglycine and D-vinylglycinol derivatives was followed by Grubbs' ring-closing metathesis to generate the key lactam intermediate. This strategy has been applied to a concise total synthesis of the potent immunostimulatory peptide FK565.

Synthesis of enantiomerically and diastereomerically pure 2(S)-amino-6(R)-hydroxy-1,7-heptanedioic acid dimethyl ester hydrochloride from cycloheptadiene.

The complete carbon framework of enantiomerically and diastereomerically pure 2(S)-amino-6(R)-hydroxy-1,7-heptanedioic acid dimethyl ester hydrochloride was derived from cycloheptadiene in six steps utilizing an amino acid-derived acylnitroso Diels-Alder reaction as the key step. This versatile amino diester has been previously used to synthesize amino-differentiated diaminopimelic acid (DAP) and biologically active analogues. In addition, after formation of a novel aminoxy diketopiperazine, the newly formed carboxyl groups were differentiated by a novel transpeptidation of the amino acid that directed the stereochemistry of the initial cycloaddition.