Nicholas A. McGrath

A Concise Ring-Expansion Route to the Compact Core of Platensimycin†

using a novel antibiotic assay approach. Characterization revealed a unique compact core connected to a highly oxygenated and unusual aromatic ring through a propionate tether. Platensimycin has a novel mechanism of action, inhibiting the b-ketoacyl-(acyl carrier protein) synthase (FabF) in the bacterial fatty acid synthetic pathway. Several new members of this class have since been reported. These differ only in functionalization of the carboxylate terminus. This attractive natural product target has also encouraged researchers to engineer strains to improve its production. Despite a flurry of synthetic activity, only two groups have completed the total syntheses of platensimycin (1). All other reported efforts have focused on constructing the platensimycin core 3. To highlight the diversity of these synthetic approaches we have chosen to emphasize the last bond formed to complete the platensimycin core as reported by each research group (Figure 2). Recently, a series of derivatives obtained by modifying platensimycin have been reported. Alternatively, several research groups have developed analogues of platensimycin, some of which were equipotent with the natural product. These results bode well for analogue approaches utilizing diverted total synthetic strategies. We envisioned a concise retrosynthetic plan for the total synthesis of platensimycin (Scheme 1). Platensic acid (2) was our immediate target as it serves later as the branch point for accessing all the other members of this natural product family. We proposed that 2 could be accessed from 4 by a retro-

Chemoselectivity in Chemical Biology: Acyl Transfer Reactions with Sulfur and Selenium

A critical source of insight into biological function is derived from the chemist’s ability to create new covalent bonds between molecules, whether they are endogenous or exogenous to a biological system. A daunting impediment to selective bond formation, however, is the myriad of reactive functionalities present in biological milieu. The high reactivity of the most abundant molecule in biology, water, makes the challenges all the more difficult. We have met these challenges by exploiting the reactivity of sulfur and selenium in acyl transfer reactions. The reactivity of both sulfur and selenium is high compared with that of their chalcogen congener, oxygen. In this Account, we highlight recent developments in this arena, emphasizing contributions from our laboratory. One focus of our research is furthering the chemistry of native chemical ligation (NCL) and expressed protein ligation (EPL), two related processes that enable the synthesis and semisynthesis of proteins. These techniques exploit the lower pKa of thiols and selenols relative to alcohols. Although a deprotonated hydroxyl group in the side chain of a serine residue is exceedingly rare in a biological context, the pKa values of the thiol in cysteine (8.5) and of the selenol in selenocysteine (5.7) often render these side chains anionic under physiological conditions. NCL and EPL take advantage of the high nucleophilicity of the thiolate as well as its utility as a leaving group, and we have expanded the scope of these methods to include selenocysteine. Although the genetic code limits the components of natural proteins to 20 or so α-amino acids, NCL and EPL enable the semisynthetic incorporation of a limitless variety of nonnatural modules into proteins. These modules are enabling chemical biologists to interrogate protein structure and function with unprecedented precision. We are also pursuing the further development of the traceless Staudinger ligation, through which a phosphinothioester and azide form an amide. We first reported this chemical ligation method, which leaves no residual atoms in the product, in 2000. Our progress in effecting the reaction in water, without an organic cosolvent, was an important step in the expansion of its utility. Moreover, we have developed the traceless Staudinger reaction as a means for immobilizing proteins on a solid support, providing a general method of fabricating microarrays that display proteins in a uniform orientation. Along with NCL and EPL, the traceless Staudinger ligation has made proteins more readily accessible targets for chemical synthesis and semisynthesis. The underlying acyl transfer reactions with sulfur and selenium provide an efficient means to synthesize, remodel, and immobilize proteins, and they have enabled us to interrogate biological systems.

Diazo compounds as highly tunable reactants in 1,3-dipolar cycloaddition reactions with cycloalkynes

Diazo compounds, which can be accessed directly from azides by deimidogenation, are shown to be extremely versatile dipoles in 1,3-dipolar cycloaddition reactions with a cyclooctyne. The reactivity of a diazo compound can be much greater or much less than its azide analog, and is enhanced markedly in polar-protic solvents. These reactivities are predictable from frontier molecular orbital energies. The most reactive diazo compound exhibited the highest known second-order rate constant to date for a dipolar cycloaddition with a cycloalkyne. These data provide a new modality for effecting chemoselective reactions in a biological context.

Diazo groups endure metabolism and enable chemoselectivity in cellulo.

We introduce a stabilized diazo group as a reporter for chemical biology. ManDiaz, which is a diazo derivative of N-acetylmannosamine, is found to endure cellular metabolism and label the surface of a mammalian cell. There its diazo group can undergo a 1,3-dipolar cycloaddition with a strained alkyne, providing a signal comparable to that from the azido congener, ManNAz. The chemoselectivity of diazo and alkynyl groups enables dual labeling of cells that is not possible with azido and alkynyl groups. Thus, the diazo group, which is approximately half the size of an azido group, provides unique opportunities for orthogonal labeling of cellular components.

An Efficient Substrate‐Controlled Approach Towards Hypoestoxide, a Member of a Family of Diterpenoid Natural Products with an Inside‐Out [9.3.1]Bicyclic Core

for generations, to treat various skin rashes and infections. Hypoestoxide has been shown in recent studies to exhibit promising anticancer, antimalarial, and anti-inflammatory activity. Our interest stems primarily from encouraging antiangiogenic activities, in which hypoestoxide was shown to inhibit the growth of a number of human and murine tumor cell lines in vivo. In terms of angiogenesis, hypoestoxide inhibited vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Hypoestoxide is a bicyclo[9.3.1]pentadecane diterpenoid containing a rigid “inside–outside” ring system decorated with an exocyclic enone, two epoxide moieties, and an acetate group. This rare ring system has also been described for the verticillanes, of which verticillol (3, Scheme 1) is the most well known. As a more oxygenated variant of verticillol, it is tempting to propose that hypoestoxide is formed from the same common cationic precursor (5) as both verticillol and taxol (4), which in turn originates from consecutive cyclizations of geranylgeranyl pyrophosphate. In the case of verticillol, the cation 5 is trapped with water, whereas, for taxol and hypoestoxide, it undergoes endocyclic and exocyclic eliminations, followed by oxygenations and cyclizations. As part of our efforts to evaluate the molecular mechanisms of promising natural product anticancer agents we have focused our investigations on hypoestoxide and the verticillanes. Several factors needed to be considered before beginning our synthetic efforts. First, for a trans-[9.3.1]bicyclic framework, two different atropisomers are possible. Calculations (B3LYP/6-311 + G(d,p)) indicated that hypoestoxide is 4.1 kcalmol 1 more stable than the atropisomer (2). Therefore, we imagined that any such macrocyclization would preferentially form the naturally occurring atropisomer. In addition, the energy change attributed to the process of interconverting hypoestoxide and the atropisomer was estimated to be 65 kcal mol . Diene 6 seemed an ideal target because it allows access to all known verticillanes, and we attempted its synthesis using a conformationally controlled ring-closing metathesis (Scheme 2). This diene provides four

An efficient oxidative dearomatization–radical cyclization approach to symmetrically substituted bicyclic guttiferone natural products

Detailed in this communication is an efficient synthetic approach towards the guttiferone family of natural products. Oxidatively unraveling a para-quinone monoketal followed by consecutive 5-exo radical cyclizations provides the bicyclic core. An additional strength of this approach is a late stage asymmetric desymmetrization of an advanced symmetric intermediate.

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

Intein-mediated expressed protein ligation (EPL) permits the site-specific chemical customization of proteins. While traditional techniques have used purified, soluble proteins, we have extended these methods to release and modify intein fusion proteins expressed on the yeast surface, thereby eliminating the need for soluble protein expression and purification. To this end, we sought to simultaneously release yeast surface-displayed proteins and selectively conjugate with chemical functionalities compatible with EPL and click chemistry. Single-chain antibodies (scFv) and green fluorescent protein (GFP) were displayed on the yeast surface as fusions to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released from the yeast surface with either a sulfur nucleophile (MESNA) or a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine azide permitted the simultaneous release and azido functionalization of displayed proteins, but nonspecific reactions with other yeast proteins were detected, and cleavage efficiency was limited. In contrast, MESNA released significantly more protein from the yeast surface while also generating a unique thioester at the carboxy-terminus of the released protein. These protein thioesters were subsequently reacted with a cysteine alkyne in an EPL reaction and then employed in an azide-alkyne cycloaddition to immobilize the scFv and GFP on an azide-decorated surface with >90% site-specificity. Importantly, the immobilized proteins retained their activity. Since yeast surface display is also a protein engineering platform, these approaches provide a particularly powerful tool for the rapid assessment of engineered proteins.

Prolyl 4-Hydroxylase: Substrate Isosteres in Which an (E)- or (Z)-Alkene Replaces the Prolyl Peptide Bond

Collagen prolyl 4-hydroxylases (CP4Hs) catalyze a prevalent posttranslational modification, the hydroxylation of (2S)-proline residues in protocollagen strands. The ensuing (2S,4R)-4-hydroxyproline residues are necessary for the conformational stability of the collagen triple helix. Prolyl peptide bonds isomerize between cis and trans isomers, and the preference of the enzyme is unknown. We synthesized alkene isosteres of the cis and trans isomers to probe the conformational preferences of human CP4H1. We discovered that the presence of a prolyl peptide bond is necessary for catalysis. The cis isostere is, however, an inhibitor with a potency greater than that of the trans isostere, suggesting that the cis conformation of a prolyl peptide bond is recognized preferentially. Comparative studies with a Chlamydomonas reinhardtii P4H, which has a similar catalytic domain but lacks an N-terminal substrate-binding domain, showed a similar preference for the cis isostere. These findings support the hypothesis that the catalytic domain of CP4Hs recognizes the cis conformation of the prolyl peptide bond and inform the use of alkenes as isosteres for peptide bonds.

Ribonucleoside 3'-phosphates as pro-moieties for an orally administered drug.

Oral administration of chemotherapeutic agents is the mainstay for the treatment of disease. Sustained release formulations have been crucial for the safe and effective dosing of orally administered drugs.[1] Such formulations allow for the prolonged maintenance of therapeutic drug concentrations, reducing the required dosages per day and thereby enhancing patient compliance. Sustained release formulations also provide tighter control over the pharmacokinetics of a drug, thereby minimizing side effects.[1] Aqueous solubility is likewise a critical attribute for an orally available drug.[2] Robust absorption across the intestinal epithelium relies upon a high concentration of the drug to drive diffusion into enterocytes and, eventually, into the circulatory system. On average, 35–40% of lead compounds have aqueous solubilities of <5 mg/mL, which is defined by the U.S. Pharmacopeia as being slightly soluble or worse.[3] Accordingly, the bioavailability and consequent efficacy of many compounds relies on enhancing their aqueous solubility.[2c] The formation of a phosphomonoester can improve the oral bioavailability of poorly water-soluble chemotherapeutic agents.[2b,2c,4] Endogenous phosphatases near the surface of enterocytes can catalyze the hydrolysis of the phosphoryl group, releasing the lipophilic drug and allowing for its efficient absorption into the body. Several prodrugs approved by the U.S. Food and Drug Administration rely on this strategy, including estramustine, fosamprenavir, and prednisolone phosphate.[4] Recently, we reported on the potential utility of a phosphodiester as the pro-moiety for a drug administered intravenously.[5] Specifically, we found that the coupling of 4-hydroxytamoxifen to uridine 3′-phosphate enabled its timed-release in serum by human pancreatic ribonuclease (RNase 1[6]; EC 3.1.27.5). This modification also increased the aqueous solubility of 4-hydroxytamoxifen. RNase 1 is an ideal endogenous enzyme to elicit pro-moiety release. A major excreted enzyme, RNase 1 has a concentration of 6.4 mg/mL in human pancreatic juice and 0.2 mg/mL in saliva, according to a radioimmunoassay.[7] Moreover, like its renowned homologue bovine pancreatic ribonuclease (RNase A[8]), RNase 1 catalyzes the cleavage of RNA by a transphosphorylation reaction[9] with little specificity for its leaving group.[10] Herein, we report on the utility of several ribonucleoside 3′-phosphates as pro-moieties for a model orally available drug, metronidazole. Metronidazole is a commonly used antibiotic for a variety of protozoa and anaerobic bacterial infections, including Bacteroides fragilis, Helicobacter pylori, Clostridium difficile, Trichomonas vaginalis, and Entamoeba histolytica.[11] In 1997, Flagyl ER, an extended release formulation of metronidazole, was approved by the FDA as a superior treatment for bacterial vaginosis. Still, metronidazole has several common side effects, such as nausea, diarrhea, and metallic taste. Moreover, metronidazole therapy can occasionally cause more severe side effects, such as pancreatitis, neutropenia, neuropathies, or CNS toxicities.[12] These adverse effects could be attenuated with better control over the pharmacokinetics of metronidazole.[13] Hence, in this proof-of-concept study, we elected to attach metronidazole to ribonucleoside 3′-phosphates to assess the attributes of this promoiety for orally available drugs (Figure 1). Figure 1 Scheme showing catalysis of the cleavage of a ribonucleoside 3′-(metronidazole phosphate) (NpMet) by RNase 1 to yield a nucleoside 2′,3′-cyclic phosphate (N>p) and Met. Each ribonucleoside 3′-(metronidazole phosphate) (NpMet) was synthesized in four steps from commercially available metronidazole (Met) and a ribonucleoside phosphoramidite (Scheme 1). Briefly, Met was coupled to the phosphoramidite by using N-methylbenzimidazolium triflate (MBIT) as a catalyst.[14] The coupled product was oxidized with iodine and deprotected stepwise. The final products were purified by chromatography on silica gel. This route was used to synthesize three different NpMets: cytidine 3′-(metronidazole phosphate) (CpMet, 18% non-optimized yield), uridine 3′-(metronidazole phosphate) (UpMet, 80%), and adenosine 3′-(metronidazole phosphate) (ApMet, 64%). Scheme 1 Route for the synthesis of NpMets. We expected the ribonucleoside 3′-phosphate moiety of an NpMet to endow the prodrug with greater hydrophilicity than the parent drug, which could improve its oral bioavailability. To investigate this issue, we calculated the partition (log P) and distribution (log D) coefficients of Met, CpMet, UpMet, and ApMet.[15] The calculated log P and log D values for the NpMets were indeed significantly lower than those of the parent drug (Table 1), indicative of increased hydrophilicity and decreased tendency to aggregate. Table 1 Calculated Partition and Distribution Coefficients of Met and NpMets[15] To be the basis for an effective timed-release prodrug strategy, the pro-moiety needs to be released by the activating enzyme over time. Hence, we used 1H NMR spectroscopy to assess the rate at which RNase 1 catalyzed the release of Met from the prodrugs (Figure S1). We assumed that pancreatic juice is diluted in the intestine, which led us to use RNase 1 at concentrations of 0.1 mg/mL and 0.01 mg/mL in these assays. Because inorganic phosphate inhibits RNase A with a Ki value of 2.3 mM,[16] we initially investigated the effect of phosphate in simulated intestinal fluid (SIF) on the rates of UpMet unmasking (Figure 2A). Compared to a buffer with no inorganic phosphate (19.5 mM Tris–HCl, pH 7.4, 2.5% v/v D2O), the rate of Met release in SIF was only marginally slower. RNase 1 cleaves after pyrimidine residues more readily than after purine residues.[6,7] Accordingly, we predicted that RNase 1 would unmask CpMet and UpMet faster than ApMet. For both concentrations of RNase 1, we did indeed observe that the cytidine and uridine prodrugs were unmasked faster than the adenosine prodrug (Figure 2B). Moreover, unlike the uridine 3′-phosphate–4-hydroxytamoxifen conjugate that cleaved spontaneously in aqueous solutions lacking ribonucleases, the NpMet conjugates were stable in SIF, which has pH 7.5, and in simulated gastric fluid (SGF), which has pH 1.1. The absence of appreciable degradation (<5%) in either medium (Figure S2) is attributable to the alkoxyl group of metronidazole being a much worse leaving group than the aryloxyl group of 4-hydroxytamoxifen. Figure 2 Progress curves for the release of Met from NpMets under various conditions as determined by 1H NMR spectroscopy. (A) Comparison of UpMet cleavage rates in Tris–HCl buffer, pH 7.4, and simulated intestinal fluid (SIF). (B) Comparison of CpMet, ... Finally, we sought to assess the antimicrobial activity of our NpMet prodrugs on B. fragilis. This penicillin-resistant Gram-negative bacillus is common in anaerobic infections, like those that originate from the gastrointestinal tract. We determined the minimum inhibitory concentration (MIC) of UpMet and ApMet, as well as Met (Figure S3). We found that both UpMet and ApMet had considerably higher MIC values than did Met (Table 2), demonstrating that the prodrugs were relatively inactive. Incubating UpMet with 0.1 mg/mL RNase 1 overnight resulted in an MIC similar to that of Met. Finally, adding UpMet to a culture medium containing RNase 1 gave an intermediate MIC, demonstrating the in situ release of the drug. Table 2 MIC Values of NpMets for Bacteroides fragilis