Discovery, Synthesis, and Optimization of Peptide-Based Antibiotics.

The rise of multidrug resistant bacteria has significantly compromised our supply of antibiotics and poses an alarming medical and economic threat to society. To combat this problem, it is imperative that new antibiotics and treatment modalities be developed, especially those toward which bacteria are less capable of developing resistance. Peptide natural products stand as promising candidates to meet this need as bacterial resistance is typically slow in response to their unique modes of action. They also have additional benefits including favorable modulation of host immune responses and often possess broad-spectrum activity against notoriously treatment resistant bacterial biofilms. Moreover, nature has provided a wealth of peptide-based natural products from a range of sources, including bacteria and fungi, which can be hijacked in order to combat more dangerous clinically relevant infections.This Account highlights recent advances in the total synthesis and development of a range of peptide-based natural product antibiotics and details the medicinal chemistry approaches used to optimize their activity.In the context of antibiotics with potential to treat Gram-positive bacterial infections, this Account covers the synthesis and optimization of the natural products daptomycin, glycocin F, and alamethicin. In particular, the reported synthesis of daptomycin highlights the utility of on-resin ozonolysis for accessing a key kynurenine residue from the canonical amino acid tryptophan. Furthermore, the investigation into glycocin F analogues uncovered a potent lead compound against Lactobacillus plantarum that bears a non-native thioacetal linkage to a N-acetyl-d-glucosamine (GlcNAc) sugar, which is otherwise O-linked in its native form.For mycobacterial infections, this Account covers the synthesis and optimization of teixobactin, callyaerin A, lassomycin, and trichoderin A. The synthesis of callyaerin A, in particular, highlighted the importance of a (Z)-2,3-diaminoacrylamide motif for antimicrobial activity against Mycobacterium tuberculosis, while the synthesis of trichoderin A highlighted the importance of (R)-stereoconfiguration in a key 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHMOD) residue.Lastly, this Account covers lipopeptide antibiotics bearing activity toward Gram-negative bacterial infections, namely, battacin and paenipeptin C. In both cases, optimization of the N-terminal lipid tails led to the identification of analogues with potent activity toward Escherichia coli and Pseudomonas aeruginosa.

The Chiral Target of Daptomycin Is the 2R,2'S Stereoisomer of Phosphatidylglycerol.

Daptomycin (dap) is an important antibiotic that interacts with the bacterial membrane lipid phosphatidylglycerol (PG) in a calcium-dependent manner. The enantiomer of dap (ent-dap) was synthesized and was found to be 85-fold less active than dap against B. subtilis, indicating that dap interacts with a chiral target as part of its mechanism of action. Using liposomes containing enantiopure PG, we demonstrate that the binding of dap to PG, the structural transition that occurs upon dap binding to PG, and the subsequent oligomerization of dap, depends upon the configuration of PG, and that dap prefers the 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol stereoisomer (2R,2'S configuration). Ent-dap has a lower affinity for 2R,2'S liposomes than dap and cannot oligomerize to the same extent as dap, which accounts for why ent-dap is less active than dap. To our knowledge, this is the first example whereby the activity of an antibiotic depends upon the configuration of a lipid head group.

A high-yielding solid-phase total synthesis of daptomycin using a Fmoc SPPS stable kynurenine synthon.

A high-yielding total synthesis of daptomycin, an important clinical antibiotic, is described. Key to the development of this synthesis was the elucidation of a Camps cyclization reaction that occurs in the solid-phase when conventionally used kynurenine (Kyn) synthons, such as Fmoc-l-Kyn(Boc,CHO)-OH and Fmoc-l-Kyn(CHO,CHO)-OH, are exposed to 20% 2-methylpiperidine (2MP)/DMF. During the synthesis of daptomycin, this side reaction was accompanied by intractable peptide decomposition, which resulted in a low yield of Dap and a 4-quinolone containing peptide. The Camps cyclization was found to occur in solution when Boc-l-Kyn(Boc,CHO)-Ot-Bu and Boc-l-Kyn(CHO,CHO)-OMe were exposed to 20% 2MP/DMF giving the corresponding 4-quinolone amino acid. In contrast, Boc-l-Kyn(CHO)-OMe was stable under these conditions, demonstrating that removing one of the electron withdrawing groups from the aforementioned building blocks prevents enolization in 2MP/DMF. Hence, a new synthesis of daptomycin was developed using Fmoc-l-Kyn(Boc)-OH, which is prepared in two steps from Fmoc-l-Trp(Boc)-OH, that proceeded with an unprecedented 22% overall yield. The simplicity and efficiency of this synthesis will facilitate the preparation of analogs of daptomycin. In addition, the elucidation of this side reaction will simplify preparation of other Kyn-containing natural products via Fmoc SPPS.

A Versatile Boc Solid Phase Synthesis of Daptomycin and Analogues Using Site Specific, On-Resin Ozonolysis to Install the Kynurenine Residue.

A de novo solid-phase synthesis of the cyclic lipodepsipeptide daptomycin via Boc chemistry was achieved. The challenging ester bond formation between the nonproteinogenic amino acid kynurenine was achieved by esterification of a threonine residue with a protected tryptophan. Subsequent late-stage on-resin ozonolysis, inspired by the biomimetic pathway, afforded the kynurenine residue directly. Synthetic daptomycin possessed potent antimicrobial activity (MIC100 =1.0 µg mL-1 ) against S. aureus, while five other daptomycin analogues containing (2R,3R)-3-methylglutamic acid, (2S,4S)-4-methylglutamic acid or canonical glutamic acid at position twelve prepared using this new methodology were all inactive, clearly establishing that the (2S,3R)-3-methylglutamic acid plays a key role in the antimicrobial activity of daptomycin.

Full solid-phase total synthesis of macrocyclic natural peptides using four-dimensionally orthogonal protective groups.

Fmoc-based solid-phase synthesis provides efficient access to both linear and macrocyclic peptides. To synthesize complex macrocyclic polyamides using Fmoc chemistry, multiple protective groups with orthogonal reactivities are generally employed because the free amines and carboxylic acids of specific residues must be selectively exposed prior to amide formation. This review focuses on four-dimensionally orthogonal protective group strategies for the full solid-phase synthesis of macrocyclic peptides with branched chains (polymyxin E2 and daptomycin) and a tricyclic natural peptide (lacticin 481).

The effect of replacing the ester bond with an amide bond and of overall stereochemistry on the activity of daptomycin.

Daptomycin, a cyclic lipodepsipeptide antibiotic, has been used clinically since 2003 to treat serious infections caused by Gram-positive bacteria. Although 37 years have passed since daptomycin's discovery, its mechanism of action is still debated. In this report, the effect of replacing the ester bond with an amide bond, and overall stereochemistry, on daptomycin's biological activity was examined. Two peptides were prepared in which the threonine4 residue in the active daptomycin analog, Dap-K6-E12-W13, was replaced with (2S,3R)-diaminobutyric acid ((2S,3R)-DABA) or its epimer (2S,3S-DABA) converting the ring-closing ester bond to an amide bond. Both of these peptides were found to be considerably less active than Dap-K6-E12-W13. These results, along with our previous studies on other daptomycin analogs, enabled us to conclude that the ester bond is crucial to daptomycin's activity. ent-Dap-K6-E12-W13 was found to be at least 133-fold less active than Dap-K6-E12-W13, indicating that a chiral interaction with a chiral target is essential to daptomycin's activity. Studies examining the binding of Dap-K6-E12-W13 and ent-Dap-K6-E12-W13 to model liposomes consisting of phosphatidylglycerol (PG) and phosphatidylcholine suggest that the stereochemistry of PG plays a crucial role in daptomycin-membrane interactions.

Structure-activity relationship of daptomycin analogues with substitution at (2S, 3R) 3-methyl glutamic acid position.

Daptomycin is a highly effective lipopeptide antibiotic against Gram-positive pathogens. The presence of (2S, 3R) 3-methyl glutamic acid (mGlu) in daptomycin has been found to be important to the antibacterial activity. However the role of (2S, 3R) mGlu is yet to be revealed. Herein, we reported the syntheses of three daptomycin analogues with (2S, 3R) mGlu substituted by (2S, 3R) methyl glutamine (mGln), dimethyl glutamic acid and (2S, 3R) ethyl glutamic acid (eGlu), respectively, and their antibacterial activities. The detailed synthesis of dimethyl glutamic acid was also reported.

α-Azido Acids in Solid-Phase Peptide Synthesis: Compatibility with Fmoc Chemistry and an Alternative Approach to the Solid Phase Synthesis of Daptomycin Analogs.

α-Azido acids have been used in solid phase peptide synthesis (SPPS) for almost 20 years. Here we report that peptides bearing an N-terminal α-azidoaspartate residue undergo elimination of an azide ion when treated with reagents that are commonly used for removing the Fmoc group during SPPS. We also report an alternative solid-phase route to the synthesis of an analog of daptomycin that uses a reduced number of α-azido amino acids and without elimination of an azide ion.

Solid-phase synthesis and in vitro biological activity of a Thr4→Ser4 analog of daptomycin.

Daptomycin is a Ca(+2)-dependent cyclic lipodepsipeptide antibiotic used clinically to treat serious infections caused by Gram-positive bacteria. The recent appearance of daptomycin-resistant strains, daptomycin's lack of activity in the presence of lung surfactant, and its incompletely understood mechanism of action underscores the need for establishing detailed structure-activity relationships. Here we report a solid-phase synthesis of a daptomycin analog in which Thr4, 3-MeGlu12 and Kyn13 in daptomycin were replaced with Ser, Glu and Trp residues, respectively (Dap-S4-E12-W13). The Thr4 to Ser4 substitution was detrimental to activity, as Dap-S4-E12-W13 was at least 20-fold less active at physiological Ca(+2) concentration than Dap-E12-W13. Much of its activity could be recovered at high (100 mM) Ca(+2) concentration, suggesting that the residue at position 4 affects Ca(+2) binding and, consequently, biological activity.

A journey to the total synthesis of daptomycin.

Daptomycin, the first antibiotic of its class, provides a new structural motif for the development of new antibiotics. Recently, we have completed the total synthesis of daptomycin. The development of the successful synthetic strategy is described here, including the application of serine/threonine ligation mediated peptide cyclization to the daptomycin macrocyclization.

Total synthesis of daptomycin by cyclization via a chemoselective serine ligation.

A total synthesis of daptomycin, the first natural product antibiotic launched in a generation, was achieved. This convergent synthesis relies on an efficient macrocyclization via a serine ligation to assemble the 31-membered cyclic depsipeptide. The difficult esterification by the nonproteinogenic amino acid kynurenine was accomplished via the esterification of a threonine residue by a suitably protected Trp ester, followed by ozonolysis. This synthesis provides a foundation and framework to prepare varied analogues of daptomycin to establish its structure-activity profile.

An efficient chemical synthesis of carboxylate-isostere analogs of daptomycin.

Herein we report a direct and efficient method for the synthesis of four new carboxylate-isostere analogs of daptomycin. The side chain carboxylic acid moieties of the aspartic acids (Asp-3, Asp-7 and Asp-9) and ß-methyl glutamic acid (MeGlu-12) were all converted into the corresponding carboxylate isosteres using direct synthetic procedures. The present study also describes an esterification protocol to overcome the possible backbone cyclization of the activated side chain carboxylic acid group of either Asp or Glu onto the backbone amide.

[Progress in the study of some important natural bioactive cyclopeptides].

Natural cyclopeptides are hot spots in chemical and pharmaceutical fields because of the wide spreading bio-resources, complex molecular structures and various bioactivities. Bio-producers of cyclopeptides distribute over almost every kingdom from bacteria to plants and animals. Many cyclopeptides contain non-coded amino acids and non-pepditic bonds. Most exciting characteristic of cyclopeptides is a range of interesting bioactivities such as antibiotics gramicidin-S (2), vancomycin (3) and daptomycin (4), immunosuppressive cyclosporin-A (1) and astin-C (8), and anti-tumor aplidine (5), RA-V (6) and RA-VII (7). Compounds 1-4 are being used in clinics; compounds 5-8 are in the stages of clinical trial or as a candidate for drug research. In this review, the progress in chemical and bioactive studies on these important natural bioactive cyclopeptides 1-8 are introduced, mainly including discovery, bioactivity, mechanism, QSAR and synthesis.

Siderophore Conjugates of Daptomycin are Potent Inhibitors of Carbapenem Resistant Strains of Acinetobacter baumannii.

Development of resistance to antibiotics is a major medical problem. One approach to extending the utility of our limited antibiotic arsenal is to repurpose antibiotics by altering their bacterial selectivity. Many antibiotics that are used to treat infections caused by Gram-positive bacteria might be made effective against Gram-negative bacterial infections, if they could circumvent permeability barriers and antibiotic deactivation processes associated with Gram-negative bacteria. Herein, we report that covalent attachment of the normally Gram-positive-only antibiotic, daptomycin, with iron sequestering siderophore mimetics that are recognized by Gram-negative bacteria, provides conjugates that are active against virulent strains of Acinetobacter baumannii, including carbapenemase and cephalosporinase producers. The result is the generation of a new set of antibiotics designed to target bacterial infections that have been designated as being of dire concern.

Engineered biosynthesis of nonribosomal lipopeptides with modified fatty acid side chains.

The biological properties of the calcium-dependent antibiotics (CDAs), daptomycin and related nonribosomal lipopeptides, depend to a large extent on the nature of the N-terminal fatty acid moiety. It is suggested that the chain length of the unusually short (C6) 2,3-epoxyhexanoyl fatty acid moiety of CDA is determined by the specificity of the KAS-II enzyme encoded by fabF3 in the CDA biosynthetic gene cluster. Indeed, deletion of the downstream gene hxcO results in three new lipopeptides, all of which possess hexanoyl side chains (hCDAs). This confirms that HxcO functions as a hexanoyl-CoA or -ACP oxidase. The absence of additional CDA products with longer fatty acid groups further suggests that the CDA lipid chain is biosynthesized on a single ACP and is then transferred directly from this ACP to the first CDA peptide synthetase (CdaPS1). Interestingly, the hexanoyl-containing CDAs retain antibiotic activity. To further modulate the biological properties of CDA by introducing alternative fatty acid groups, a mutasynthesis approach was developed. This involved mutating the key active site Ser residue of the CdaPS1, module 1 PCP domain to Ala, which prevents subsequent phosphopantetheinylation. In the absence of the natural module 1 PCP tethered intermediate, it is possible to effect incorporation of different N-acyl-L-serinyl N-acetylcysteamine (NAC) thioester analogues, leading to CDA products with pentanoyl as well as hexanoyl side chains.

Depsipeptide synthesis.

Naturally occurring cyclic depsipeptides, peptides that contain one or more ester bonds in addition to the amide bonds, have emerged as an important source of pharmacologically active compounds or promising lead structures for the development of novel synthetically derived drugs. This class of natural products has been found in many organisms, such as fungi, bacteria, and marine organisms. It is very well known that cyclic depsipeptides and their derivatives exhibit a diverse spectrum of biological activities, including insecticidal, antiviral, antimicrobial, antitumor, tumor-promotive, anti-inflammatory, and immunosuppressive actions. However, they have shown the greatest therapeutic potential as anticancer and particularly antimicrobial agents. Difficulties associated with isolation and purification of larger quantities of this class of natural products and, particularly, unlimited access to their synthetic analogs significantly hampered cyclic depsipeptides exploitation as lead compounds for development of new drugs. As an alternative, total solution or solid-phase peptide synthesis of these important natural products and combinatorial chemistry approaches can be employed to elucidate structure-activity relationships and to find new potent compounds of this class. In this chapter, methods for formation of depsipeptide ester bonds, hydroxyl group protection, and solid-phase reaction monitoring are described.

An Acyl-Linked Dimer of Daptomycin Is Strongly Inhibited by the Bacterial Cell Wall.

The lipopeptide antibiotic daptomycin is active against Gram-positive pathogens. It permeabilizes bacterial cell membranes, which involves the formation of membrane-associated oligomers. We here studied a dimer of daptomycin whose two subunits were linked through a bivalent aliphatic acyl chain. Unexpectedly, the dimer had very low activity on vegetative Staphylococcus aureus and Bacillus subtilis cells. However, activity resembled that of monomeric daptomycin on liposomes and on B. subtilis L-forms. These findings underscore the importance of the bacterial cell wall in daptomycin resistance.

Fluorescein-labeled Bacitracin and Daptomycin Conjugates: Synthesis, Fluorescence Imaging and Evaluation.

BACKGROUND: Previously, glycopeptides antibiotics such as vancomycin, ramoplanin and an antifungal antibiotic nystatin have been studied for their diagnostic and therapeutic potential. OBJECTIVE: To further explore the diagnostic and chemotherapeutic potential of other antibiotics we have now employed daptomycin, a lipopetide antibiotic and bacitracin, a polypeptide antibiotic in uptake and vitality tests on human cell lines. METHOD: Fluorescent conjugates of bacitracin and daptomycin were synthesized using fluorescein isothiocynate (FITC) for confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS). The cellular uptake of the synthesized daptomycin and bacitracin conjugates was studied on seven human cell lines, two healthy and five malignant using CLSM and FACS. To examine the cell membrane damage caused by the conjugates FACS experiments were carried out using propidium iodide. RESULTS: The uptake pattern was different for both antibiotics for all the cell lines. The cytoplasmic uptake of daptomycin conjugate was lower than the bacitracin conjugate, resulting in decreased cell membrane damage. CONCLUSION: No preferential uptake into malignant or healthy cells was found for the two different antibiotic conjugates and the uptake patterns were also different between the two antibiotics. However, the lower cytotoxicity and different uptake mechanism makes daptomycin conjugate a prospective candidate for further study as a diagnostic agent for various intracellular infections.

Solid-phase total synthesis of daptomycin and analogs.

An entirely solid-phase synthesis of daptomycin, a cyclic lipodepsipeptide antibiotic currently in clinical use, was achieved using a combination of α-azido and Fmoc amino acids. This methodology was applied to the synthesis of several daptomycin analogs, one of which did not contain kynurenine or the synthetically challenging amino acid (2S,3R)-methylglutamate yet exhibited an MIC approaching that of daptomycin.