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Bioorthogonal Chemistry - Requirements X The Living Cell Living cells are a complex network of interacting biopolymers, ions, and metabolites. Complex cellular processes cannot be observed when the components are in their purified, isolated forms. Thus, we are forced outside of the artificial confines of a test tube and into the dynamic living cell. There is a burgeoning interest in developing methods where a reporter tag can be attached to a cellular component for aid in visualization and/or isolation. Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13-21. David Goodshell, artist and biologist X X Bioorthogonal Chemistry - Requirements water as the solvent ambient temperature non-toxic reagents physiological pH reaction product must be stable no cross-reactivity cell-permeable reagents fast reaction kinetics Sletten, E. M.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6974-6998. X X A Brief Consideration of Reaction Kinetics A B A B most bioorthogonal reactions are bimolecular in nature with second-order rate constants -3 3 -1 -1 [product] is approximately k2 [A]0[B]0t k2 is typically anywhere from 10 to 10 M s -1 -1 k2 = 2.3 M s , [A] = [B] = 1 µM, time = 1 hr ~ 0.8 % labeling of product Sletten, E. M.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6974-6998. Lim, R. K. V.;Lin, Q. Chem. Commun. 2010, 46, 1589-1600. X X Biosynthesis of mucin-type O-linked glycoproteins OH OH OH O HO O HO HO OH NH NH OH O O Me Me N-Acetylegalactosamine N-Acetylglucosamine GalNAc GlcNAc GalNAc GlcNAc 1-kinase salvage pathway OH OH OH O HO O O HO NH HO NH O O O NH O O -2 P P N OPO3 O O Me O O O O Me HO OH GalNAc-1-P UDP-GlcNAc Hang, H. C.; Yu, C.; Kato, D.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2003, 100, 14846-14851. Dube, D. H.; Prescher, J. A.; Quang, C. N.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2006, 103, 4819-4824. X X Biosynthesis of mucin-type O-linked glycoproteins GalNAc-1-P UDP-GlcNAc UDP-GalNAc UDP-Glc/GlcNAc pyrophosphorylase C4-epimerase OH OH O O HO NH NH O O O O P P N O O Me O O O O HO OH UDP-GalNAc Hang, H. C.; Yu, C.; Kato, D.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2003, 100, 14846-14851. Dube, D. H.; Prescher, J. A.; Quang, C. N.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2006, 103, 4819-4824. X X Bioorthogonal Chemistry R'' O N condensation NH2 chemistry R'' R R' R R' O O Staudinger OMe N3 NHR ligation R PPh2 P(O)Ph2 N R N N [3+2] cycloadditions N3 R R' R' olefin chemistry RS RS X X Bioorthogonal Chemistry R'' O N condensation NH2 chemistry R'' R R' R R' O O Staudinger OMe N3 NHR ligation R PPh2 P(O)Ph2 N R N N [3+2] cycloadditions N3 R R' R' olefin chemistry RS RS X X Condensation Chemistry ! Unnatural amino acid mutagenesis was performed on T4 lysozyme incorporated at two solvent accessible sites - Ser44 and Ala82 CO2H Me CO2H O NH2 O O NH2 Me 5% efficiency 30% efficiency determined by catalytic activity, SDS-PAGE, and autoradiography Cornish, V. W.; Hahn, K. M.; Schultz, P. G. J. Am. Chem. Soc. 1996, 118, 8150-8151. Zhang, Z.; Smith, B. A. C.; Wang, L.; Brock, A.; Cho, C.; Schultz, P. G. Biochemistry 2003, 42, 6735-6746. X X Condensation Chemistry ! Cell surface remodeling O Me OH - HO HN OH CO2 O O HO O HO H O HO OH O N HO ManLev Me O a sialic acid analogue O HN NH biotin O S 4 OH NH CO - R NHNH2 O O OH 2 5 HO O H O Hydrazide HN N N HO Me O Keppler, O. T.; Horstkorte, R.; Pawlita, M.; Schmidts, C.; Reutter, W. Glycobiology 2001, 11, 11R- 18R. Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science 1997, 276, 1125-1128. X ManNAc. We selected three human cell el of fluorescence, suggesting that unnat- by sialidase digestion, as quantified by lines—Jurkat (T cell– derived), HL-60 ural sialosides are the major biosynthetic high pH anion exchange chromatography (neutrophil-derived), and HeLa (cervical products of ManLev. (HPAEC), was approximately 10 times epithelial carcinoma)—to test the biosyn- Ideally, proof of the cell surface expres- lower than that released from ManNAc- thetic conversion of ManLev to the cor- sion of ketone-bearing sialic acids would treated cells (13, 20). responding cell surface–associated, unnat- involve abrogating the fluorescence signal There are two possible explanations for ural sialic acid (Fig. 1A). Cells were treat- by treatment with sialidase enzymes. How- the observed reduction in normal sialic acid ed with ManLev, and the presence of ke- ever, the commercially available sialidases on ManLev-treated cells: (i) normal sialic tone groups on the cell surface was were found to be inactive against N-levu- acid is replaced with the unnatural sialic determined by the chemoselective ligation linoyl sialosides, in accordance with their acid during incubation with ManLev, or (ii) of a hydrazide-based probe, biotinamido- known reduced activity against sialosides the biosynthesis of all sialosides is sup- caproyl hydrazide (Fig. 1B). The cells were with other unnatural N-acyl groups (19). pressed during incubation with ManLev. analyzed by flow cytometry after being We therefore evaluated the effects of Inhibition of sialoside biosynthesis would stained with FITC (fluorescein isothiocya- ManLev treatment on the amount of nor- cause an increase in exposed terminal ga- nate) avidin (15). The Jurkat, HL-60, and mal sialic acid on Jurkat cells, expecting a lactose residues, the penultimate residue in HeLa cells treated with ManLev showed a reduction. Indeed, the amount of sialic the majority of sialoglycoconjugates. We large increase in fluorescence intensity acid released from ManLev-treated cells therefore examined the effect of ManLev compared to cells treated with buffer or the natural derivative ManNAc (Fig. 2). ManLev-treated cells that were stained with FITC-avidin alone, without prior bi- otinamidocaproyl hydrazide treatment, showed only a background fluorescence. These results indicate that ManLev-treat- ed cells express cell surface–associated ke- tone groups and can be chemoselectively decorated with hydrazide conjugates even in the presence of serum. on March 7, 2012 We performed a series of experiments to demonstrate that the ketone groups are dis- played on the cell surface in the form of modified sialoglycoconjugates. Jurkat cells were treated with tunicamycin, an inhibitor of N-linked protein glycosylation, before in- Fig. 1. Biosynthetic incorpora- cubation with ManLev (16,X17). We antic- tion of ketone groups into cell ipated a dramatic reduction in ketone ex- surface–associated sialic acid. (A) N-Levulinoyl mannosamine pression on the basis of the observation that Condensation Chemistry www.sciencemag.org most mature (and therefore sialylated) oligo- (ManLev, ML) is metabolically converted to the corresponding cell surface sialoside. (B)Cells saccharides on Jurkat cells are found on N- displaying ketone groups can be chemoselectively ligated to hydrazides under physiological condi- tions through the formation of an acyl hydrazone. Cell surface ketones were conjugated to biotinami- linked rather than O-linked glycop!ro t eCinesll surface remodeling (18). Indeed, ketone expression resulting docaproyl hydrazide to provide a tag for subsequent detection with FITC-avidin. In principle, any hydrazide-derivatized molecule can be used to selectively remodel the surface of ketone-expressing from ManLev treatment was inhibited by cells. tunicamycin in a dose-dependent fashion (Fig. 3A), suggesting that the ketone groups are presented on oligosaccharides and are Downloaded from not nonspecifically associated with cell sur- face components. In contrast, ketone expres- sion in HL-60 and HeLa cells was unaffected by tunicamycin, but was instead blocked by a-benzyl N-acetylgalactosamine, an inhibi- tor of O-linked glycosylation, consistent with the high expression of mucin-like mol- ecules on myeloid- and epithelial-derived cell lines. Although we predicted that ManLev would be converted into the correspond- ing sialoside, we addressed the possibility that ketone expression resulted from con- version of ManLev to N-levulinoyl glu- cosamine (GlcLev) by the enzyme that interconverts ManNAc and GlcNAc (12). Fig. 2. Ketone expression in Jurkat, HL-60, and HeLa cells. Cells treated with buffer alone or with In that GlcNAc is incorporated into most ManNAc showed only background fluorescence. Cells treated with ManLev showed up to a 30-fold glycoproteins, GlcLev might have many increase in fluorescence above background. The fluorescence increase was dependent on both bio- avenues for cell surface expressiocne.llFsl owweretinamidocaproyl analyzed by hydrazide flow cy (Bio)tom andetry FITC-avidin after sta (Av)inin treatment.g with FI ResultsTC (fl wereuore similarscein in is threeothi replicateocyanate) avidin cytometry revealed only a background lev- experiments for each cell line. 1126 SCIENCE z VOL. 276 z 16 MAY 1997 z www.sciencemag.org Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science 1997, 276, 1125-1128. X X Condensation Chemistry tunicamycin HO O N HO NH alpha-benzyl N-acetylglucosamine O O HO OH HO O HO Me HO HO O NH O Me O NH O Me O O NHAc HO HO OH Addition of tunicamycin, a known inhibitor of N-linked protein glycosylation, inhibits ketone expression with ManLev treatment in Jurkat cells. Ketone expression was blocked on HL-60 and HeLa cells via alpha-benzyl N-acetylgalactosamine, an inhibitor of O-linked glycosylation. Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science 1997, 276, 1125-1128. X X Condensation Chemistry ! Selective drug delivery? H N Biotin O N O O Me Me H2NHN Biotin drug drug avidin S H avidin H N Biotin N Biotin S N S N S O O Me Me selective drug delivery Mahal, L.
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