The Living Cell. Living cells are a complex network of interacting biopolymers, ions, and metabolites.

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2 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.. at. Chem. Biol. 2005, 1, David Goodshell, artist and biologist

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6 Bioorthogonal Chemistry - equirements 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.. Angew. Chem. Int. Ed. 2009, 48,

7 A Brief Consideration of eaction Kinetics A B A B most bioorthogonal reactions are bimolecular in nature with second-order rate constants [product] is approximately k 2 [A] 0 [B] 0 t k 2 is typically anywhere from 10-3 to 10 3 M -1 s -1 k 2 = 2.3 M -1 s -1, [A] = [B] = 1 µm, time = 1 hr ~ 0.8 % labeling of product Sletten, E. M.; Bertozzi, C.. Angew. Chem. Int. Ed. 2009, 48, Lim,. K. V.;Lin, Q. Chem. Commun. 2010, 46,

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10 Biosynthesis of mucin-type -linked glycoproteins H H H H H H Me -Acetylegalactosamine GalAc H H H Me H -Acetylglucosamine GlcAc GalAc 1-kinase GlcAc salvage pathway H H H H H Me P 3-2 H H H Me P P H H H GalAc-1-P UDP-GlcAc Hang, H. C.; Yu, C.; Kato, D.; Bertozzi, C.. Proc. atl. Acad. Sci. USA 2003, 100, Dube, D. H.; Prescher, J. A.; Quang, C..; Bertozzi, C.. Proc. atl. Acad. Sci. USA 2006, 103,

11 Biosynthesis of mucin-type -linked glycoproteins GalAc-1-P UDP-GlcAc UDP-GalAc pyrophosphorylase UDP-Glc/GlcAc C 4 -epimerase H H H H Me P P H H H UDP-GalAc Hang, H. C.; Yu, C.; Kato, D.; Bertozzi, C.. Proc. atl. Acad. Sci. USA 2003, 100, Dube, D. H.; Prescher, J. A.; Quang, C..; Bertozzi, C.. Proc. atl. Acad. Sci. USA 2006, 103,

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13 Bioorthogonal Chemistry condensation chemistry '' H 2 ' '' ' Staudinger ligation PPh 2 Me 3 H P()Ph 2 [3+2] cycloadditions 3 olefin chemistry S ' S '

14 Bioorthogonal Chemistry condensation chemistry '' H 2 ' '' ' Staudinger ligation PPh 2 Me 3 H P()Ph 2 [3+2] cycloadditions 3 olefin chemistry S ' S '

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17 Condensation Chemistry! Unnatural amino acid mutagenesis was performed on T4 lysozyme incorporated at two solvent accessible sites - Ser 44 and Ala 82 Me H 2 C 2 H Me H 2 C 2 H 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, Zhang, Z.; Smith, B. A. C.; Wang, L.; Brock, A.; Cho, C.; Schultz, P. G. Biochemistry 2003, 42,

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19 Condensation Chemistry! Cell surface remodeling H H H H H Me H H H H H C 2 - ManLev a sialic acid analogue Me H H biotin HH 2 Hydrazide S 4 H 5 H H H H H H C 2 - Me Keppler,. T.; Horstkorte,.; Pawlita, M.; Schmidts, C.; eutter, W. Glycobiology 2001, 11, Mahal, L. K.; Yarema, K. J.; Bertozzi, C.. Science 1997, 276,

20 Condensation Chemistry! Cell surface remodeling Fig. 2. cells were analyzed by flow cytometry after staining with FITC (fluorescein isothiocyanate) avidin Mahal, L. K.; Yarema, K. J.; Bertozzi, C.. Science 1997, 276,

21 Condensation Chemistry tunicamycin H H H alpha-benzyl -acetylglucosamine Me Me H H H H H H HAc H H H H H Me Addition of tunicamycin, a known inhibitor of -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 -acetylgalactosamine, an inhibitor of -linked glycosylation. Mahal, L. K.; Yarema, K. J.; Bertozzi, C.. Science 1997, 276,

22 Condensation Chemistry! Selective drug delivery? H Biotin Me Me H 2 H Biotin drug H Me Biotin avidin S S drug H Me avidin Biotin S S selective drug delivery Mahal, L. K.; Yarema, K. J.; Bertozzi, C.. Science 1997, 276,

23 Condensation Chemistry! Selective drug delivery? toxicity of the conjugate was dependent on the expression of ketones cells with high ketone expression (~700,000 ketones per cell) were sensitive to lethal doses of ricin LD 50 between 1 to 10 nm ricin inhibits protein synthesis cells with low ketone expression (~50,000 ketones per cell) showed no toxicity indicates the potential for cell surface engineering to support selective drug delivery Mahal, L. K.; Yarema, K. J.; Bertozzi, C.. Science 1997, 276,

24 Bioorthogonal Chemistry condensation chemistry '' H 2 ' '' ' Staudinger ligation PPh 2 Me 3 H P()Ph 2 [3+2] cycloadditions 3 olefin chemistry S ' S '

25 Azide - A Powerful Chemical eporter absent from biological systems possesses orthogonal reactivity to most biological functional groups the azide is small, so biological perturbation is minimal first used as a chemical reporter in 2000 in the Staudinger Ligation Griffin,. J. Prog. Med. Chem. 1994, 31, 121. Hendricks, S. B.; Pauling, L. J. Am. Chem. Soc. 1925, 47, Saxon, E.; Bertozzi, C.. Science 2000, 287, 2007.

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29 The Staudinger Ligation Jurkat cells were incubated with azidoacetylmannosamine at a concentration 20 mm for three days. Cell viability was tested with incubation for up to six days. Cells were washed and reacted with phosphine for 1 hour at a concentration of 1 mm. Stained with FITC-avidin and analyzed by flow cytometry. Saxon, E.; Bertozzi, C.. Science 2000, 287, 2007.

30 The Staudinger Ligation Biological Controls - Could a Staudinger reduction be taking place and the phosphine oxide localizes in or outside the cell? Could the phosphine be reducing disulfide bonds and not be completely bioorthogonal? H H P H TCEP Saxon, E.; Bertozzi, C.. Science 2000, 287, 2007.

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32 Activation of a Fluorgenic Dye via the Staudinger Ligation The lone pair of electrons on phosphorus quench the excited fluorophore coumarin dye C 2 Me P 3 Me H CH 3 C/H 2 Me H H P Ph not fluorescent strongly fluorescent 3 C 2 H H 2 L - azidohomoalanine Phosphine dye was reacted with recombinant murine dihydrofolate reductase bearing azidohomoalanine residues. Unnatural amino acids incorporated during overexpression in a methionine auxotrophic E. coli strain. Fluorescence was observed over background. o washing or Western blotting necessary. Lemieux, G. A.; de Graffenried, C. L.; Bertozzi, C.. J. Am. Chem. Soc. 2003, 125,

33 Activation of a Fluorgenic Dye via the Staudinger Ligation A small problem P C 2 Me [] Me 2 C P Ph not fluorescent strongly fluorescent xidation of the probe by air would provide background fluorescence. Hangauer, M. J.; Bertozzi, C.. Angew. Chem. Int. Ed. 2008, 47, 2394.

34 Activation of a Fluorgenic Dye via the Staudinger Ligation Fluorescence resonance energy transfer (FET) based probe FET 2 H no fluorescence PPh 2 2 Me quencher 3, H 2 H 2 H PPh 2 + H Me fluorescence Hangauer, M. J.; Bertozzi, C.. Angew. Chem. Int. Ed. 2008, 47, 2394.

35 "Traceless" Staudinger Ligation Peptide coupling by the traceless Staudinger ligation 2 3 Peptide 2 Peptide 1 S PPh 2 S Peptide 1 PPh 2 Peptide 2 Peptide 1 H Peptide 2 H 2 Peptide 1 Peptide 2 PPh 2 Ph 2 P SH SH ilsson, B. L.; Kiessling, L. L.; aines,. T. rg. Lett. 2000, 2, Soellner, M. B.; ilsson, B. L.; aines,. T. J. Am. Chem. Soc. 2006, 128, 8820.

36 "Traceless" Staudinger Ligation Traceless Staudinger ligation is reminiscent of native chemical ligation Peptide 1 S H 2 HS Peptide 2 thioesterification Peptide 1 H 2 S Peptide 2 S- acyl shift Peptide 1 H SH Peptide 2 Dawson, P. E.; Muir, T. W.; Clarklewis, I.; Kent, S. B. H. Science 1994, 266, 776.

37 Staudinger Ligation Staudinger ligation PPh 2 Me 3 H P()Ph 2 The Staudinger ligation has been used to probe biomolecules within living animals. Second-order kinetics where the rate-determining step is purported to be attack of the phosphine on the azide. k = 10-3 M -1 s -1 (very slow) High concentrations of the phosphine are often necessitated (> 250 um) Attempts to increase phosphine nucleophilicity has increased the suspectibility of phosphine oxidation. Lin, F. L.; Hoyt, H. M.; Van Halbeek, H.; Bergman,. G.; Bertozzi, C.. J. Am. Chem. Soc. 2005, 127, Prescher, J. A.; Dube, D. H.; Bertozzi, C.. ature 2004, 430, 873.

38 Bioorthogonal Chemistry condensation chemistry '' H 2 ' '' ' Staudinger ligation PPh 2 Me 3 H P()Ph 2 [3+2] cycloadditions 3 olefin chemistry S ' S '

39 [3 + 2] Cycloadditions ' 3 ' First discovered by Arthur Michael in In the 1950s, olf Huisgen proposed that the reaction proceeds through a 1,3-dipolar cycloaddition. High temperatures and pressure required made this reaction largely impractical. In separate efforts, Meldal and Sharpless reported the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC). Ph Ph CuS 4. 5H 2 1 mol% sodium ascorbate 5% H 2 :tbuh, T Ph Ph 91% ostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int. Ed. 2002, 41, Tornoe, C. W.; Christensen, C.; Meldal, M. J. rg. Chem. 2002, 67, 3057.

40 [3 + 2] Cycloadditions CuL n-1 ' ' 1,4-disubstituted 1,2,3-triazoles copper(iii) metallacycle CuL n-2 ' [L n Cu] + Catalytic Cycle H alkyne CuL n-2 ' CuL n-1 copper acetylide ' azide Himo, F.; Lovell, T.; Hilgraf,.; ostovtsev, V. V.; oodleman, L.; Sharpless, K. B.; Fokin, V. V. J. Am. Chem. Soc. 2005, 127,

41 Copper(I) Toxicity Ph Ph CuS 4. 5H 2 1 mol% sodium ascorbate 5% H 2 :tbuh, T Ph Ph 91% Finn, Sharpless, and coworkers reported the first biomolecule coupling application of [3+2] cycloaddition through the attachment of dyes to the cowpea mosaic virus. CuAAC is not widely employed, due to copper(i)'s toxicity. E. Coli stops dividing after exposure to 100 um CuBr for 16 hours. Mammalian cells and zebrafish embryos can survive low concentrations of copper (I) (< 500 um) but considerable cell death is observed above 1 mm. Wang, Q.; Chan, T..; Hilgraf,.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, Chan, T..; Hilgraf,.; Sharpless, K. B.; Fokin, V. V. rg. Lett. 2004, 6, Link, A. J.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, Speers, A. E.; Adam, B. F.; Cravatt, B. F. J. Am. Chem. Soc. 2003, 125, 4686.

42 [3+2] Cycloaddition - ing Strain Strain-promoted azide cycloadditions were first investigated by Alder and Stein in the 1930s. In the 1960s, Krebs and Wittig commented that phenylazide and cyclooctyne "proceeded like an explosion" massive bond angle deformation of the acetylene to 163 o ~ 18 kcal/mol of ring strain Wittig, G.; Krebs, A. Chem. Ber. 1961, 94, Alder, K.; Stein, G. Justus Liebigs Ann. Chem. 1931, 485, 211.

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45 In Vivo Imaging of Zebrafish Embryos H F F DIF 3 copper-free click chemistry Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C.. Science 2008, 320, 664.

46 In Vivo Imaging of Zebrafish Embryos First confirmed that zebrafish glycan biosynthetic enzymes are tolerant of the unnatural sugar. Zebrafish cell line ZF4 was incubated with various doses of Ac 4 GalAzm reacted with DIF-488 and analyzed by flow cytometry. Azide-labeled cell lysates were further characterized by treatment with a DIF-Flag peptide conjugate. bserved high-molecular weight species were consistent with labeled glycoproteins. Flag-containing species (glycoproteins like b-hexosaminidase, b-integrin, nicastrin) were known or predicted sites of mucin-type -linked glycosylation. ig. 2. In vivo imaging of Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C.. Science 2008, 320, 664. m on March 7, 2012

47 In Vivo Imaging of Zebrafish Embryos From 60 hours post-fertilization (hpf) to 72 hpf, a burst in fluorescence intensity in the jaw region, pectoral fins, and olfactory organs. These three areas are reexamined and reacted with DIF-647 between 60 and 61 hpf and DIF-488 between 61 and 62 hpf. mouth region pectoral fin jaw region olfactory region Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C.. Science 2008, 320, 664.

48 In Vivo Imaging of Zebrafish Embryos Three-dye bioimaging - DIF-647 between 60 and 61 hpf, DIF-488 between 62 and 62 hpf, and then DIF-555 between 72 and 73 hpf. jaw region olfactory region cells kinocilia Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C.. Science 2008, 320, 664.

49 DIF Synthesis H 1) ah 2) Br H PCC 52% 91% H cat. LHMDS, 99%, 1:1 1) LHMDS, - 78 o C 2) TESCl 91% SiEt 3 F + F Selectfluor 95% 1.0:2.2 Baskin, J. M.; Prescher, J. A.; Laughlin, J. T.; Agard,. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C.. Proc. atl. Acad. Sci. USA 2007, 104,

50 DIF Synthesis F 1) KHMDS, - 78 o C 2) TESCl TES F Selectfluor F F 97% 4:1 desired:undesired 74% ucl 3 ai 4 93% F F LDA, 0 o C Tf F F 1) KHMDS, - 78 o C 2) Tf 2 Ph F F H 2 C 11% H 2 C 47% H 2 C Baskin, J. M.; Prescher, J. A.; Laughlin, J. T.; Agard,. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C.. Proc. atl. Acad. Sci. USA 2007, 104,

51 Cyclooctyne Analogues F Me HC F H Me HC DIF, Bertozzi Lab k = M -1 s -1 BAAC, Bertozzi Lab k = 0.96 M -1 s -1 DIMAC, Bertozzi Lab more water soluble k = M -1 s -1 H Bu Boons and Popik Labs photocaged k = M -1 s -1 H DIB, Boons Lab 5 steps nontoxic, k = M -1 s -1 Sletten, E. M.; Bertozzi, C.. rg. Lett. 2008, 10, ing,. H.; Guo, J.; Wolfert, M. A.; Boons, G. J. Angew. Chem. Int. Ed. 2008, 47, Sletten, E. M.; Bertozzi, C.. Acc. Chem. es. 2009, 44,

52 Bioorthogonal Chemistry condensation chemistry '' H 2 ' '' ' Staudinger ligation PPh 2 Me 3 H P()Ph 2 [3+2] cycloadditions 3 olefin chemistry S ' S '

53 Inverse-Electron Demand Diels-Alder - 2 H H k 2 = 2000 M -1 s -1 thioredoxin Trx SH Trx S Trx S H H H H Blackman, M. L.; oyzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008, 130,

54 Inverse-Electron Demand Diels-Alder - 2 H H k 2 = 2000 M -1 s -1 Trx = Da Michael product = Da Diels-Alder product = Da Blackman, M. L.; oyzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008, 130,

55 Inverse-Electron Demand Diels-Alder H CD 3 D H H H H H H k 2 = 22,000 M -1 s -1 trans-cyclooctene ring is designed via computation (M06L/6-311+G(d,p)-optimized transition structure) Taylor, M. T.; Blackman, M. L.; Dmitrenko,.; Fox, J. M. J. Am. Chem. Soc. 2011, 133,

56 Inverse-Electron Demand Diels-Alder H CD 3 D H H H H H H k 2 = 22,000 M -1 s -1 trans-cyclooctene ring is designed via computation (M06L/6-311+G(d,p)-optimized transition structure) Taylor, M. T.; Blackman, M. L.; Dmitrenko,.; Fox, J. M. J. Am. Chem. Soc. 2011, 133,

57 Inverse-Electron Demand Diels-Alder H CD 3 D H H H H H H k 2 = 22,000 M -1 s -1 trans-cyclooctene ring is designed via computation (M06L/6-311+G(d,p)-optimized transition structure) Taylor, M. T.; Blackman, M. L.; Dmitrenko,.; Fox, J. M. J. Am. Chem. Soc. 2011, 133,

58 Inverse-Electron Demand Diels-Alder Genetically encoded norbornene directs site-specific cellular protein labelling + r.t. aqueous H rthogonal synthetase/ta pair used to install a norbornene-containing amino acid Me H H H 2 Pyrrolysine "The 22nd Amino Acid" H Genetically coded amino acid used by some methanogenic archaea. Pyrrolysyl-tA synthetase/ta CUA is orthogonal to endogenous tas and aminoacyl-ta synthetases in E. coli and eukaryotic cells. Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin, J. W. ature Chem. 2012, Advanced nline Publication.

59 Inverse-Electron Demand Diels-Alder Genetically encoded norbornene directs site-specific cellular protein labelling + r.t. aqueous H rthogonal synthetase/ta pair used to install a norbornene-containing amino acid H HBoc H H 2 :MeH (95:5) HBoc H 21 o C H H k = 9 M -1 s -1 Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin, J. W. ature Chem. 2012, Advanced nline Publication.

60 Inverse-Electron Demand Diels-Alder Genetically encoded norbornene directs site-specific cellular protein labelling + r.t. aqueous H The fluorescence of certain probes increased by a 5-10 fold increase after the cycloaddition H H H 2 "turn-on" fluorophorogenic probes - tetrazine can quench fluorophore via energy transfer tetramethylrhomdamine (TAMA) Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin, J. W. ature Chem. 2012, Advanced nline Publication.

61 Inverse-Electron Demand Diels-Alder Cycloaddition tested with mutated epidermal growth factor receptor (EGF-GFP) 2 (1 mm) egfp TAMA Merged + DIC H 2 H CH 3 (1 mm) egfp TAMA Merged + DIC H 2 H Me CH Me Me Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin, J. W. ature Chem. 2012, Advanced nline Publication.

62 Photochemical 1,3-Dipolar Cycloaddition 310 nm 10 minutes 2 fluorescent 10-3 to 10-1 M -1 s -1 + Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew. Chem. Int. Ed. 2008, 47, Wang, Y.; Hu, W. J.; Song, W.; Lim,. K. V.; Lin, Q. rg. Lett. 2008, 10, Song, W.; Wang, Y.; Lin, Q. J. Am. Chem. Soc. 2008, 130, 9654.

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65 Future utlook Groups 15 elements have been particularly lucrative. Perhaps larger elements in this group, like bismuth or antimony, may be of use. Pericyclic reactions have been very promising as well, due to concerted mechanisms that leave little room for interruption from other components. Applications with other sources of energy, such as light or ultrasound, may occur. We may see the extension of chemical reporters to other small-molecule metabolites. Sletten, E. M.; Bertozzi, C.. Angew. Chem. Int. Ed. 2009, 48,

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