Post Glycosylation Diversification (PGD): An Approach for Assembling Collections of Glycosylated Small Molecules

Zachary Cannone, a Ala Shaqra,b Chris Lorenc,a Liza Henowitz, b Santosh Keshipeddy,c Victoria Robinson,b Adam Zweifach,b Dennis Wright,a,c Mark W. Peczuha* aDepartment of Chemistry, University of Connecticut, 55 N. Eagleville Road U3060, Storrs, CT bDepartment of Molecular & Cellular Biology, University of Connecticut, 91 N. Eagleville Road U3125, Storrs, CT cDepartment of Pharmaceutical Sciences, School of Pharmacy, 69 N. Eagleville Road U3092, University of Connecticut, Storrs, Connecticut 06269

Abstract: Many small molecule natural products with antibiotic and antiproliferative activity are adorned with a carbohydrate residue as part of their molecular structure. The carbohydrate moiety can act to mediate key interactions with the target, attenuate physicochemical properties, or both. Facile incorporation of a carbohydrate group on de novo small molecules would enable these valuable properties to be leveraged in the evaluation of focused compound libraries. While there is no universal way to incorporate a sugar on small molecule libraries, techniques such as glycorandomization and neoglycorandomization have made signification headway toward this goal. Here we report a new approach for the synthesis of glycosylated small molecule libraries. It puts the glycosylation early in the synthesis of library compounds. Functionalized aglycones subsequently participate in chemoselective diversification reactions distal to the carbohydrate. As a proof-of-concept we prepared a number of desosaminyl glycosides from only a few starting glycosides, using click cycloadditions, acylations, and Suzuki couplings as diversification reactions. New compounds were then characterized for their inhibition of bacterial protein translation, bacterial growth, and in a T-cell activation assay.

INTRODUCTION Removal of glucose by a glycosidase, which is Carbohydrate moieties on glycosylated concomitant with export, weaponizes the small molecules play specific roles that . Sugar residues on , attenuate their biological activities.1,2,3 In another macrolide inhibitor of translation, can addition to their influence on physicochemical be beneficial or detrimental to its antibiotic properties, sugar residues often serve as activity, depending on the context. The handles that mediate binding interactions cladinose sugar of erythromycin acts as a between the small molecule and its target, handle used by the efflux pumps that confer and underpin their associated activity. macrolide resistance to bacteria. Efflux out of Glycosylated natural products4 epitomize this the bacterial cytoplasm prevents association phenomenon, where the sugars on macrolide with the ribosome, and therefore a loss of antibiotics are illustrative. The producer of the activity. In fact, so called third generation bacterial translation inhibitor methymycin, for like telithromycin lack the example, adopts a pro-drug strategy using a cladinose residue by design so that the glucose moiety (Fig. 1).5 When glucose is compound is a poor substrate for efflux.6,7 attached to the desosamine residue of the Methymycin, erythromycin, and telithromycin macrolide, the compound does not fit into the all contain a desosamine sugar. Desosamine binding site in the ribosome exit tunnel. makes contacts with the RNA near the ribosomal exit tunnel that are essential to its edge of glycosylated small molecule library activity. Specifically, there is a H-bond synthesis.16,17,18,19 Enzymes from glycosylated between its C2 hydroxyl and the N1 of natural product biosynthetic clusters are adenine 2058 (E. coli) in the ribosome and an mixed and matched, or mutated to relax electrostatic interaction between the substrate specificity in the glycorandomization protonated C3 dimethylamino group and a approach. Neoglycorandomization is a nearby phosphate.8,9 These macrolide chemical approach that leverages the examples additionally underscore the chemoselective reaction between an N,O- importance of amino sugars in particular as disubstituted alkoxyamine unit with a reducing mediators of small molecule bioactivity, sugar to yield a neoglycoside. An alternative especially in the context of small molecule – to “diversify then glycosylate” is “glycosylate nucleic acid interactions. then diversify.” The idea is to prepare a small collection of key glycosylated precursors that O O serve as feedstocks in library assembly. The HO HO feedstock compounds, containing reactive OH desosamine O desosamine handles on the aglycone, can be subsequently HO N(CH ) O O O O O 3 2 elaborated at sites distal from the glycosyl O O N(CH ) O O 3 2 O domain. Through the careful choice of HO OCH3 OH precursor-reaction pairs, it should be possible HOHO O OH to assemble highly focused libraries through glucose cladinose short, flexible and protecting group-free Methymycin Erythromycin sequences. We refer to this method for the Figure 1. Methymycin and Erythromycin structures preparation of glycosylated small molecule highlighting their important sugar moieties. libraries as post-glycosylation diversification,

or PGD. Here we report a proof-of-concept The advantages of incorporating PGD study using desosamine as the key carbohydrates onto lead molecules are sugar for library synthesis. New glycosides attractive for the development of new drug 10,11,12,13 prepared by this method were then candidates. A traditional approach characterized for their potential activity as toward the synthesis of families of antibiotic and immunomodulatory lead glycosylated small molecules follows a compounds. “diversify then glycosylate” strategy. In such a scenario, an aglycone library is synthesized RESULTS AND DISCUSSION and parallel glycosylations are conducted Desosamine 1 (Scheme 1) was chosen as afterward to complete the library assembly. the residue upon which we would build The glycosylation reactions used to prepare glycosylated small molecule libraries as a library compounds, however, are notorious for proof-of-concept demonstration of PGD. The 14,15 being temperamental. More precisely, main factors that influenced this choice were each new substrate can require slightly the ready availability of desosamine from different reaction conditions and reagents that erythromycin via hydrolysis, and the fact that need to be optimized to affect reaction. amino sugars frequently contribute to binding Moreover, the glycosylations can give rise to when they are present on small molecules. A anomeric mixtures that are difficult to separate well-established sequence of reactions was by chromatography. These drawbacks make used to form the starting material for PGD the preparation of libraries through parallel precursor preparation.20 Desosamine was glycosylation daunting. Recent methods, converted to its pyrimidyl glycoside via a specifically glycorandomization and Mitsunobu reaction with mercaptopyrimidine, neoglycorandomization, represent the cutting and then the C2 hydroxyl group was protected as its methyl carbonate to give compound 2 Reaction mixtures were loaded onto a silica (49%, over two steps). Initially, three different column that had been pre-treated with alcohols were glycosylated with this donor: triethylamine where nonpolar impurities were azido ethanol, m-bromobenzyl alcohol, and p- washed off with hexanes and products were bromobenzyl alcohol. After glycosylation, the eluted by increasing the polarity of the mobile carbonate was removed by solvolysis in a phase with ethyl acetate. Further, methanol water mixture. Yields for the two- cycloaddition reactions showed a propensity step sequence are shown in Scheme 1. to go to near completion, with yields in the Azido-ethyl desosaminyl glycoside 3 was also 80% range, leaving few impurities to remove converted to the aminoethyl species via from the crude reaction mixtures. Amide and catalytic hydrogenation to give compound 4 sulfonamide analogs were also prepared from (82 %). The four new desosaminyl glycosides, aminoethyl glycoside 4 via acylation reactions. 3 – 6, were then poised for diversification by Using an amino functionalized PGD precursor cycloaddition, acylation/sulfonylation, and was logical because it was accessible from Pd(0) mediated coupling reactions, as the azido compound and it could be diversified described in the following paragraphs. directly to yield amides or sulfonamides, respectively. Treatment of the amines with Scheme 1. Synthesis of PGD precursors from acid chlorides in the presence of a base gave desosamine N rise to amide products 10a-c, in varying yields O OH 1) mercaptopyrimidine, O S (Table 2). Sulfonamide 10d was prepared DIAD, PBu3 N OH 2) methyl chloroformate, from amine 4 and phenylsulfonyl chloride in a NaHCO , THF OCO2CH3 N(CH3)2 3 N(CH3)2 similar manner. Purifications were carried out 49% (two steps) 1 2 in the same manner as other diversification sequences with normal phase = 1) alcohol, AgOTf, chromatography using gradient elution. O mol. sieves, 0°C N3 H , 2 3 49% (from 2) 2) CH3OH/H2O Pd/C 80°C Table 1. Cycloaddition reactions with PGD O precursor 3 H2N

4 49% (from 3) O O R1 R2 O O N O O N N 3 N CuSO4, ascorbate O Br OH OH OH t-BuOH, H2O R1 R2 N(CH ) N(CH3)2 N(CH3)2 5 (meta) 58% (from 2) 3 2

6 (para) 65% (from 2) 3 - 6 Yield R1 R2 Cmpd. (%) We began our diversification efforts on azidoethyl and aminoethyl precursors 3 and 4. H 7a 81

Azidoethyl PGD precursor 3 was diversified via simple cycloaddition click reactions using N 21 different alkynes (Table 1). Using H 7b 60 established procedures,22 we coupled 3 to CN substituted alkynes with Cu(II) and catalytic ascorbic acid in aqueous alcohol. Use of 8 commercially available phenyl acetylene gave OCH3 triazole 7a (Table 1). Additionally, propyargylic 23 biphenyl compounds such as 8 and 9 gave H OCH3 7c 82 rise to 7b and 7c, respectively. Purification of the products was facilitated by their tendency N to be retained on normal phase silica gel. 9 substitution, or heteroatoms on the aromatics Table 2. Acylation reactions with PGD precursor 4 attached onto the aglycone.

O O acyl/sulfonyl chloride O O R Because the motivation behind our NH2 N NaOH H development of PGD was to build libraries for OH OH THF, H2O screening campaigns, we sought assays that N(CH3)2 N(CH3)2 could be used to evaluate the new Yield compounds. Protein translation was a logical R Cmpd. (%) starting point in a search for potential activity O because the desosamine moiety in the initial 10a 54 PGD library is present on antibacterial translation inhibitory macrolides such as O erythromycin, , , 10b 12 C 2 etc. It was likely our compounds would have H O some affinity to the negatively charged rRNA 10c 6 under physiological pH which would render the amine protonated. We considered it O O S unlikely that the small library would 10d 61 recapitulate the activity of those macrocycles.

Rather, we hoped to find compounds that The extensive scope and reliability of might inhibit protein translation, but were palladium catalyzed coupling reactions guided structurally simpler than erythromycin itself. us towards the Suzuki reaction as an Importantly, compounds with these qualities additional avenue for diversifying desosamine. would be amenable to development in terms Palladium mediated couplings tend to play a of medchem to increase potency and tune prominent role in medchem campaigns and physicochemical properties. In vitro reliably allow for the incorporation of aromatic transcription-translation assays have been and heteroaromatic moieties that can established primarily for the small-scale modulate target binding. A common set of synthesis of new proteins. The assay also has reaction conditions were identified24 and then been used to identify inhibitors of this 25 utilized to prepare the compounds in Table 3. process. There is significant potential for The procedure entailed heating a mixture of new compounds that can stop, or stall, protein the appropriate desosaminyl glycoside translation. Stalling has been the primary containing an aryl bromide on its aglycone mechanism of action of the macrolides as 26 (i.e, 5 or 6) and boronic acid/boronic ester in antibacterials. Recently, a selective, small the presence of Cs2CO3 and Pd(Cl)2(PPh3)2 in molecule moldulator of the human ribosome 27 a dioxane/water mixture. Purification of as it translates PCSK9 was identified. This products in this series was once again exciting result opens the possibility of facilitated by their tendency to be retained identifying other modulators of human under normal phase silica chromatography translation. PGD compound libraries, like the with low polarity solvents. Increasing the one reported here, could be candidates for polarity eluted the compounds with little such studies. contamination. The meta-substituted benzyl glycoside 5 yielded biaryl analogs 11a-f. Similarly, para-substituted glycoside 6 gave rise to biaryls 12a-f; yields were largely consistent over the couplings that were conducted. The boronic acids/esters were chosen to vary the size, electronics, Table 3. Suzuki reactions with PGD precursors 5 and 6

Br B(OH)2 O O O O R X R X , Cs2CO3

OH Dioxane, H2O OH N(CH3)2 N(CH3)2

R(meta) Cmpd. Yield (%) R(para) Cmpd. Yield (%)

11a 75 12a 45

11b 54 12b 52

N 11c 20 12c 41 N N

11d 29 12d 55 N N H F 11e 39 O 12e 42 N O 11f 15 12f 37 O O solution without vector was subtracted from Experimentally, the PURExpress cell-free each emission scan. Inhibition is reported transcription/translation system (New England relative to the control reaction with vector and Biolabs, Inc) was adapted so that DMSO. Two compounds, 11a and 11b, fluorescence would provide a quantitative showed some inhibition in an earlier version of estimate of protein synthesis based on the the cell-free transcription/translation assay signal produced from maturing emerald green where DHFR production was monitored by fluorescent protein, EmGFP.25 characteristic PAGE (data not shown).28 In the fluorescence fluorescence emission was observed in the assay, compound 11c demonstrated weak absence of vector, indicating that the signal in inhibition (Fig. 2D), reducing the EmGFP the assay came primarily from EmGFP rather signal by about one-third (35%). Erythromycin, than being intrinsic to the PURExpress kit included as a positive control, reduced (Fig. 2A). Although there were slight variations fluorescence by 99% at 1 μg/mL (Fig. 2F), between PURExpress lots used in the study, suggesting almost complete inhibition of addition of 175-400 ng of vector DNA to the EmGFP synthesis. reaction mixture consistently yielded a robust Analogs of 11 that varied the linker portion signal. Addition of vector to the assay initiated of the aglycone were also synthesized (Fig. the biosynthesis of EmGFP and produced a 3). The connection between the desosamine continuous increase in fluorescence intensity moiety and the “linker” aromatic ring was at 507 nm that reached a plateaued after shortened (13), lengthened (14), or modified three or four hours (Fig. 2B, 2C). Library to contain an additional hydroxymethyl group compounds 7 and 10-12 were then tested for (15). When the new compounds were their ability to inhibit EmGFP production at a evaluated in the cell-free single concentration (25 μg/mL in DMSO), transcription/translation fluorescence assay, where total DMSO concentration was <1% by compound 15 showed greater inhibition than volume. To normalize measurements, 11b or 11c, reducing of EmEFG fluorescence background fluorescence signals of the assay by approximately 70% (Fig. 2E). In order to

Figure 2. (A-C) Cell free expression of EmGFP using the PURExpress kit: (A) Emission spectra obtained from reactions with 200 ng pRSET plasmid or without plasmid. (B) Optimization of vector amount and (C) assay time to achieve the highest fluorescence signal in the reactions. (D-F) Fluorescence emission profiles for PUREXpress assays done in the presence of desosamine compounds. Reactions were performed as describe in the materials and methods section. Fluorescence signal when the expression was run in the presence of: (D) 25 μg/mL compound 11c. (E) 25 μg/mL compound 15. (D) 1 μg/mL Erythromycin.

rule out that the possibility that the inhibitors were false positives due to fluorescence O O quenching, we incubated purified EmGFP with O O OH increasing concentrations of the inhibitory N(CH3)2 OH compound. No fluorescence quenching was N(CH3)2 detected, even at inhibitor concentrations 13 14 above 1 mM. Compounds investigated in this study did not possess intrinsic fluorescence activity in isolation when excited at 480 nm (See Supporting Information). Compound 15 O O OH also inhibited bacterial cell growth, with an MIC ≤50 μg/mL against S. aureus 29213, K. OH N(CH3)2 pneumoniae 10031,and E. coli strains 15 NB6900 and 25922. The data suggest Figure 3. Desosaminyl glycoside analogs 13-15. inhibition of protein synthesis as a possible mechanism of action for 15. While we have The collection of compounds (7, 10-15) was not confirmed it experimentally, it is also screened for activity in an assay that reasonable to postulate that the ribosome, monitors binding of a fluorescent antibody to a with its highly conserved macrolide binding lysosomal protein (LAMP-1) that is transferred pocket, is the target for the desosaminyl to the cells’ plasma membrane upon lytic glycosides described here. granule exocytosis. Because the assay is conducted using flow cytometry, it is not necessary to wash away unbound antibody. To initiate the assay, TALL-104 human method. It leverages the investment of an leukemic cytotoxic T cells are stimulated with early stage glycosylation reaction toward a beads coated with anti-CD3 antibodies.29 pay-off that delivers individual compounds Compound 14 inhibited exocytosis in a time- through straightforward, chemoselective dependent manner, with > 75% inhibition at a reactions in library synthesis. A variety of concentration of 10 µM after 24 hour reactive handles, in addition to those incubation. It does not appear to affect cell demonstrated here, should be amenable to viability, assessed via the percentage of cells the PGD approach.30,31 A potential drawback in the live cell forward scatter vs side scatter is that unprotected sugars, which are the gate. preferred PGD precursors, may be poorly Because this assay measures the final soluble in organic solvents or give rise to output of the upstream signaling pathway that unwanted side products under the conditions controls the process, it has the ability to detect of a given diversification reaction. Many click compounds that 1) inhibit TCR-proximal reactions, however, tolerate aqueous reaction signals; 2) inhibit plasma membrane calcium conditions, or they can be optimized to channels; 3) inhibit plasma membrane overcome solubility and selectivity challenges. potassium channels (which help maintain the Compounds from our small PGD library driving force for calcium entry; 4) inhibit showed weak inhibition of protein synthesis in calmodulin; 5) inhibit calcineurin catalytic an in vitro transcription-translation assay using activity; 6) inhibit MAPKK; 7) inhibit ERK; or 8) E. coli ribosomes. Inhibitory compounds also inhibit PKC. In our previous work using TCR- showed antibacterial activity again Gram independent chemical stimulation, we positive and Gram negative bacteria. Also, identified 160 compounds in the NIH’s one library member inhibited T-cell activation MLSMR that blocked lytic granule exocytosis. in a cell-based assay. The assay results Among the 75 that were available for demonstrate two pathways that are enabled retesting, we found compounds that inhibited by access to PGD libraries. In one, sugars Ca2+ influx, and thus could be Ca2+ channel or with known binding interactions with a target K+ channel blockers, compounds that inhibited can be leveraged as an anchoring fragment in MAPKK activity, and a compound that a discovery campaign. In the other, PGD inhibited calcineurin activity and therefore library compounds are screened in high could be either a calmodulin or calcineurin content assays in a target agnostic fashion. In inhibitor. We were able to exclude the either scenario, further SAR or medchem of possibility that any compounds that we found interesting compounds should be tractable that affected lytic granule exocytosis acted on based on the strategy used for their synthesis. PKC or on ERK catalytic activity. The assay also provides a rough measure of compound ACKNOWLEDGMENTS effects on cell viability by assessing changes Parts of this work were supported by a in light scatter. Research Excellence Program (REP) grant from the Office of the Vice President for CONCLUSION Research at the University of Connecticut. PGD is a viable platform for assembling glycosylated small molecule libraries that can AUTHOR INFORMATION be evaluated for activity in biochemical and *Corresponding Author biological screens. Late-stage diversifications Email: [email protected] on the aglycones of functionalized desosaminyl glycosides via cyloadditions, ORCID: acylations, and Suzuki couplings reported Mark W Peczuh: 0000-0002-3028-6551 here constitute proof-of-concept of the PGD REFERENCES 547–555. (13) Walvoort, M. T. C.; Lukose, V.; Imperiali, B. A Modular Approach to (1) Meutermans, W.; Le, G. T.; Becker, B. Phosphoglycosyltransferase Inhibitors Inspired Carbohydrates as Scaffolds in Drug Discovery. by Nucleoside Antibiotics. Chem. - A Eur. J. ChemMedChem 2006, 1 (11), 1164–1194. 2016, 22 (11), 3856–3864. (2) Thanh Le, G.; Abbenante, G.; Becker, B.; (14) Christensen, H. M.; Oscarson, S.; Jensen, H. H. Grathwohl, M.; Halliday, J.; Tometzki, G.; Zuegg, Common Side Reactions of the Glycosyl Donor J.; Meutermans, W. Molecular Diversity through in Chemical Glycosylation. Carbohydr. Res. Sugar Scaffolds. Drug Discov. Today 2003, 8 2015, 408, 51–95. (15), 701–709. (15) Ranade, S. C.; Demchenko, A. V. Mechanism of (3) Weymouth-Wilson, A. C. The Role of Chemical Glycosylation: Focus on the Mode of Carbohydrates in Biologically Active Natural Activation and Departure of Anomeric Leaving Products. Nat. Prod. Rep. 1997, 14 (2), 99. Groups. J. Carbohydr. Chem. 2013, 32 (1), 1– (4) Elshahawi, S. I.; Shaaban, K. A.; Kharel, M. K.; 43. Thorson, J. S. A Comprehensive Review of (16) Gantt, R. W.; Peltier-Pain, P.; Thorson, J. S. Glycosylated Bacterial Natural Products. Chem. Enzymatic Methods for Soc. Rev. 2015, 44 (21), 7591–7697. Glyco(Diversification/Randomization) of Drugs (5) Lishan Zhao, ‡; Noelle J. Beyer, ‡; Svetlana A. and Small Molecules. Nat. Prod. Rep. 2011, 28 Borisova, ‡,§ and; Hung-wen Liu*, ‡,§. β- (11), 1811. Glucosylation as a Part of Self-Resistance (17) Kren, V.; Rezanka, T. Sweet Antibiotics - the Mechanism in Methymycin/ Role of Glycosidic Residues in Antibiotic and Producing Strain Streptomyces Venezuelae†. Antitumor Activity and Their Randomization. 2003. FEMS Microbiol. Rev. 2008, 32 (5), 858–889. (6) Llano-Sotelo, B.; Dunkle, J.; Klepacki, D.; (18) Yu, H.; Chen, X. One-Pot Multienzyme (OPME) Zhang, W.; Fernandes, P.; Cate, J. H. D.; Systems for Chemoenzymatic Synthesis of Mankin, A. S. Binding and Action of CEM-101, a Carbohydrates. Org. Biomol. Chem. 2016, 14 New Fluoroketolide Antibiotic That Inhibits (10), 2809–2818. Protein Synthesis. Antimicrob. Agents (19) Goff, R. D.; Thorson, J. S. Neoglycosylation and Chemother. 2010, 54 (12), 4961–4970. Neoglycorandomization: Enabling Tools for the (7) Bryskier, A. Ketolides—Telithromycin, an Discovery of Novel Glycosylated Bioactive Example of a New Class of Antibacterial Agents. Probes and Early Stage Leads. Med. Chem. Clin. Microbiol. Infect. 2000, 6 (12), 661–669. Commun. 2014, 5 (8), 1036–1047. (8) Dunkle, J. A.; Xiong, L.; Mankin, A. S.; Cate, J. (20) Breton, P.; Hergenrother, P. J.; Hida, T.; H. D. Structures of the Escherichia Coli Hodgson, A.; Judd, A. S.; Kraynack, E.; Kym, P. Ribosome with Antibiotics Bound near the R.; Lee, W.-C.; Loft, M. S.; Yamashita, M.; et al. Peptidyl Transferase Center Explain Spectra of Total Synthesis of Erythromycin B. Tetrahedron Drug Action. Proc. Natl. Acad. Sci. U. S. A. 2007, 63 (26), 5709–5729. 2010, 107 (40), 17152–17157. (21) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click (9) LeTourneau, N.; Vimal, P.; Klepacki, D.; Mankin, Chemistry: Diverse Chemical Function from a A.; Melman, A. Synthesis and Antibacterial Few Good Reactions. Angew. Chemie Int. Ed. Activity of Desosamine-Modified Macrolide 2001, 40 (11), 2004–2021. Derivatives. Bioorg. Med. Chem. Lett. 2012, 22 (22) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; (14), 4575–4578. Sharpless, K. B. A Stepwise Huisgen (10) Zuegg, J.; Muldoon, C.; Adamson, G.; Cycloaddition Process: Copper(I)-Catalyzed McKeveney, D.; Le Thanh, G.; Premraj, R.; Regioselective “Ligation” of Azides and Terminal Becker, B.; Cheng, M.; Elliott, A. G.; Huang, J. Alkynes. Angew. Chemie Int. Ed. 2002, 41 (14), X.; et al. Carbohydrate Scaffolds as 2596–2599. Glycosyltransferase Inhibitors with in Vivo (23) G-Dayanandan, N.; Scocchera, E. W.; Antibacterial Activity. Nat. Commun. 2015, 6 (1), Keshipeddy, S.; Jones, H. F.; Anderson, A. C.; 7719. Wright, D. L. Direct Substitution of Arylalkynyl (11) Moukha-chafiq, O.; Reynolds, R. C. Parallel Carbinols Provides Access to Diverse Terminal Solution-Phase Synthesis and General Acetylene Building Blocks. Org. Lett. 2017, 19 Biological Activity of a Uridine Antibiotic Analog (1), 142–145. Library. ACS Comb. Sci. 2014, 16 (5), 232–237. (24) G-Dayanandan, N.; Paulsen, J. L.; Viswanathan, (12) Tang, L.; McDaniel, R. Construction of K.; Keshipeddy, S.; Lombardo, M. N.; Zhou, W.; Desosamine Containing Polyketide Libraries Lamb, K. M.; Sochia, A. E.; Alverson, J. B.; Using a Glycosyltransferase with Broad Priestley, N. D.; et al. Propargyl-Linked Substrate Specificity. Chem. Biol. 2001, 8 (6), Antifolates Are Dual Inhibitors of Candida Albicans and Candida Glabrata. J. Med. Chem. 2014, 57 (6), 2643–2656. (25) Rosenblum, G.; Chen, C.; Kaur, J.; Cui, X.; Goldman, Y. E.; Cooperman, B. S. Real-Time Assay for Testing Components of Protein Synthesis. Nucleic Acids Res. 2012, 40 (12), e88–e88. (26) Kannan, K.; Kanabar, P.; Schryer, D.; Florin, T.; Oh, E.; Bahroos, N.; Tenson, T.; Weissman, J. S.; Mankin, A. S. The General Mode of Translation Inhibition by Macrolide Antibiotics. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (45), 15958–15963. (27) Lintner, N. G.; McClure, K. F.; Petersen, D.; Londregan, A. T.; Piotrowski, D. W.; Wei, L.; Xiao, J.; Bolt, M.; Loria, P. M.; Maguire, B.; et al. Selective Stalling of Human Translation through Small-Molecule Engagement of the Ribosome Nascent Chain. PLOS Biol. 2017, 15 (3), e2001882. (28) Harvey, C. J. B.; Puglisi, J. D.; Pande, V. S.; Cane, D. E.; Khosla, C. Precursor Directed Biosynthesis of an Orthogonally Functional Erythromycin Analogue: Selectivity in the Ribosome Macrolide Binding Pocket. J. Am. Chem. Soc. 2012, 134 (29), 12259–12265. (29) Zhao, Z.; Haynes, M. K.; Ursu, O.; Edwards, B. S.; Sklar, L. A.; Zweifach, A. A High-Throughput Phenotypic Screen of Cytotoxic T Lymphocyte Lytic Granule Exocytosis Reveals Candidate Immunosuppressants. J. Biomol. Screen. 2015, 20, 359–372. (30) Becer, C. R.; Hoogenboom, R.; Schubert, U. S. Click Chemistry beyond Metal-Catalyzed Cycloaddition. Angew. Chemie Int. Ed. 2009, 48 (27), 4900–4908. (31) Beal, D. M.; Jones, L. H. Molecular Scaffolds Using Multiple Orthogonal Conjugations: Applications in Chemical Biology and Drug Discovery. Angew. Chemie Int. Ed. 2012, 51 (26), 6320–6326.