Bioorthogonal Chemistry - Requirements

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

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 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

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 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-treated cells express cell surface–associated ketone 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 displayed 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 conditions 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 surface components. In contrast, ketone expression in HL-60 and HeLa cells was unaffected by tunicamycin, but was instead blocked by a-benzyl N-acetylgalactosamine, an inhibitor 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 corresponding sialoside, we addressed the possibility that ketone expression resulted from conversion 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. K.; Yarema, K. J.; Bertozzi, C. R. Science 1997, 276, 1125-1128. X X 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

LD50 between 1 to 10 nM

cells with low ketone expression (~50,000 ketones per cell) showed no toxicity ricin inhibits protein synthesis indicates the potential for cell surface engineering to support selective drug delivery

Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science 1997, 276, 1125-1128. 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 Azide - A Powerful Chemical Reporter

N N N

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, R. J. Prog. Med. Chem. 1994, 31, 121. Hendricks, S. B.; Pauling, L. J. Am. Chem. Soc. 1925, 47, 2904. Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007. X

R E P O R T S edly with the extensive biotinylation of azido- abound inside cells in the form of metabolites olized to the glucuronidated compound, without sugar-treated cells reacted with phosphine 5 such as pyruvic acid and oxaloacetate. The significant reduction (14). (Fig. 5A). We conclude that the chemoselective modified Staudinger reaction is chemically or- The delivery of azides to cell surfaces ligation reaction proceeds as designed without thogonal to ketone ligations and should allow through other carbohydrate biosynthetic path- complications arising from nonspecific amine tandem modification of cell surfaces with the ways could significantly expand applications acylation. two chemistries. of cell surface engineering. Azides and phos- To satisfy the requirement of chemical or- The susceptibility of azides to reduction phines are abiotic structures both inside and thogonality, both participants in the reaction during the metabolic process warrants some outside cells, which raises the exciting possi- may not engage functional groups endogenous consideration in light of the reducing potential bility that their ligation could proceed in the to cells. Triarylphosphines are mild reducing of the cell’s interior. Monothiols such as gluta- intracellular environment. Given existing agents, which raises the the possibility of disul- thione can reduce alkyl azides at alkaline pH, powerful methods for incorporating unnatural fide bond reduction as an undesirable side re- but the rates of such reactions under physiolog- building blocks into other biopolymers, one action. We addressed this issue by incubating ical conditions are insignificant on the time need not be restricted to cell surface oligo- Jurkat cells with triarylphosphine 4, an interme- scale of our experiments (13). Correspondingly, saccharides as hosts for these chemical han- diate in the synthesis of 5, and quantifying the metabolic studies of the azido drug AZT dles (15, 16). Azido–amino acids, for exam- appearance of free sulfhydryl groups on the cell (azidothymidine) showed 90% recovery of the ple, could be introduced into proteins and surface with iodoacetylbiotin and FITC-avidin azide, either in its administered form or metab- later targeted with phosphine probes. The (Fig. 5B). After 1 hour in the presence of 1 mM 4, no detectable increase in free sulfhydryl groups was observed relative to cells exposed to Fig. 5. Specificity of the modified Staudinger iodoacetylbiotin alone. In a positive control ex- reaction. (A) Cell surface biotinylation does not proceed by classical Staudinger azide reduction periment, we incubated Jurkat cells with the followed by nonspecific acylation. Jurkat cells trialkylphosphine TCEP (1 mM, 1 hour), a were cultured in the presence of acetylated 3 commercial disulfide bond reducing agent. A as described in Fig. 4. Cell surface azides were marked increase in free cell-surface sulfhydryl either reduced intentionally with a trisulfon- groups was observed in this case (Fig. 5B). We ated triphenylphosphine or left unreduced. conclude that triarylphosphines such as 4 and 5 Phosphine oxide 6, the product of the classical on March 7, 2012 X Staudinger reaction, was prepared indepen- are essentially unreactive toward disulfide dently and incubated with the cells (1 mM for bonds under these conditions, rendering liga- 1 hour). Analysis by flow cytometry was per- tion with azides the predominant pathwTay hfore Staudinger Ligation formed as in Fig. 4. (B) Triarylphosphines do not reactivity. reduce disulfide bonds at the cell surface. Jurkat In a side-by-side comparison with our pre- cells were incubated with a 1 mM solution of viously reported cell surface ketone reaction triarylphosphine 4 or TCEP for 1 hour at room temperature. The cells were centrifuged (2 min, (2), the ceJllusrukrafatc eceSltlasu dwinegreer pinroccuesbsawteasd with azidoacetylmannosamine at a conc3e0n00trag)t,iowna s2he0d mwMith PBS, and diluted to a superior infosre vtehrarleere sdpeacytss. .Using the same volume of 240 ml. Samples were combined with reagent concentrations, azidosugar metabolism 60 ml of a solution of iodoacetylbiotin (5 mM in www.sciencemag.org followed by phosphine reaction produced two- PBS). After incubation in the dark at room fold higherCfleuollr evsciaenbcielitthya nwkaesto stuegsatremde twabi-th incubation for up to six days. temperature for 1.5 hours, the cells were olism followed by hydrazide reaction. This may washed with buffer, stained with FITC-avidin, and analyzed by flow cytometry. In both (A) reflect either a faster reaction at the cell surface and (B), error bars represent the standard de- or more effCicieenlltsm weteabroeli swmaosfhazeido saungadr 3reasacted with phosphine for 1 hour at a concveianttiorantoiof ntw oofr e1p lmicaMte.experiments. compared with ketosugar 2 (Fig. 1). The azide has a major advantage over the ketone in that its reactivity isSutnaiiqnueedin wa ictehll uFlaIrTcCon-atevxtidoiwni nagnd analyzed by flow cytometry. Downloaded from to its abiotic nature. Ketones, by contrast,

Fig. 4. (A) Analysis of cell surface reaction by flow cytometry. Jurkat cells (1.25 3 105 cells per milliliter) were cultured in the presence or ab- sence (control) of acetylated 3 (20 mM for 3 days). The cells were washed twice with 1 ml of buffer (0.1% fetal bovine serum in PBS, pH 7.4) and diluted to a volume of 240 ml. Samples were added to 60 ml of a solution of 5 (5 mM in PBS, pH 7.4) and incubated at room temperature for 1 hour. The cells were washed and re- suspended in 100 ml of buffer, then added to 100 ml of FITC-avidin staining Soabxtaoine, dEi.n; Btweortroezplzici,a tCe .e Rxp.e Srimcieenntsc.e(B 2) 0P0ro0g,r e2ss87o,f 2re0a0ct7io.n over time. solution (1:250 dilution in PBS). After a 10-min incubation in the dark at Assays were performed as in (A) with 40 mM acetylated 3 and varying the 4°C, the cells were washed with 1 ml of buffer and the FITC-avidin staining duration of the reaction with compound 5. (C) pH profile of reactioXn. Assays was repeated. The cells were washed twice with buffer, then diluted to a were performed as in (A) with 40 mM acetylated 3 and varying the pH of the volume of 300 ml for flow cytometry analysis. Similar results were buffer used during incubation with compound 5.

www.sciencemag.org SCIENCE VOL 287 17 MARCH 2000 2009 R E P O R T S edly with the extensive biotinylation of azido- abound inside cells in the form of metabolites olized to the glucuronidated compound, without sugar-treated cells reacted with phosphine 5 such as pyruvic acid and oxaloacetate. The significant reduction (14). (Fig. 5A). We conclude that the chemoselective modified Staudinger reaction is chemically or- The delivery of azides to cell surfaces ligation reaction proceeds as designed without thogonal to ketone ligations and should allow through other carbohydrate biosynthetic path- complications arising from nonspecific amine tandem modification of cell surfaces with the ways could significantly expand applications acylation. two chemistries. of cell surface engineering. Azides and phos- To satisfy the requirement of chemical or- The susceptibility of azides to reduction phines are abiotic structures both inside and thogonality, both participants in the reaction during the metabolic process warrants some outside cells, which raises the exciting possi- may not engage functional groups endogenous consideration in light of the reducing potential bility that their ligation could proceed in the to cells. Triarylphosphines are mild reducing of the cell’s interior. Monothiols such as gluta- intracellular environment. Given existing agents, which raises the the possibility of disul- thione can reduce alkyl azides at alkaline pH, powerful methods for incorporating unnatural fide bond reduction as an undesirable side re- but the rates of such reactions under physiolog- building blocks into other biopolymers, one action. We addrRessEedP tOhisR iTssuSe by incubating ical conditions are insignificant on the time need not be restricted to cell surface oligo- edly wXith the extensive biotinylation of azido- Jaubrokuantdceilnlssiwdeithcetrllisariynlpthoesfpohrimneo4f, maneitnabteorlmites- oscliazledotfootuhreegxlpuecruirmoneindtast(e1d3c).oCmoproreusnpdo,nwdiitnhgoluyt, saccharides as hosts for these chemical han- sugar-treated cells reacted with phosphine 5 dsiuacthe iansthpeyrsuyvnitcheasicsidofa5n,danodxaqluoancetitfaytien.gTthe smigentaifbiocalinct rsetduudcietisono(f14th).e azido drug AZT dles (15, 16). Azido–amino acids, for exam- (Fig. 5A). We conclude that the chemoselective ampopdeaifriaendcSetoafudfrienegesurlrfehaycdtrioynl girsocuhpesmoincathlleycoerl-l (aziTdohtehydmeliidvinerey) sohfowaezdid9e0s%torecceolvlersyurofafctehse ple, could be introduced into proteins and ligation reaction proceeds as designed without stuhorTfgaochneawel itto hSkieotdtoonaaecuelitgydalbtiioointnisnagannddesFhrIoT uCLld-aaivlgildoiwnatitaohzriodneu,gehitohtehreirnciatrsbaodhmydinriastteerbeidosfyonrmtheotricmpeatathb-- later targeted with phosphine probes. The complications arising from nonspecific amine (tFanigd.e5mB)m. Aodftiefirc1athioonuroifncthelel psruersfeanccees owfi1thmthMe ways could significantly expand applications acylation. 4tw, onochedmetiesctrtaiebsl.e increase in free sulfhydryl of cell surface engineering. Azides and phos- Fig. 5. Specificity of the modified Staudinger To satisfy the requirement of chemical or- grouTphsewsaussocbespetribvieldityreloaftivaeztiodecselltsoexrepdouscetdioton phines are abiotic structures both inside and iodoacetylbiotin alone. In a positive control ex- reaction. (A) Cell surface biotinylation does not thogonality, both partBiciipoanlotsginictahle Creoacntitornolsd -uring the metabolic process warrants some outside cells, which raises the exciting possi- proceed by classical Staudinger azide reduction may not engage functional groups endogenous pceornimsideenrta,tiwone iinncluigbhatteodf tJhuerkraetducceilnlsg wpoithentihael bility that their ligation could proceed in the followed by nonspecific acylation. Jurkat cells to cells. Triarylphosphines are mild reducing torifatlhkeylcpehlol’sspihnitneerioTr.CMEPono(1thimolMs s,uc1h hasougrl)u,taa- intracellular environment. Given existing were cultured in the presence of acetylated 3 commercial disulfide bond reducing agent. A as described in Fig. 4. Cell surface azides were agents, which raises theCtoheupldos saib iSlityaoufddiinsugl-er rtehdiounectciaonnr ebdeuc etaaklkiynlga zpidlaescaet alknadli ntehpeH p, hopsopwheirnfuel moextihdoeds lfoocr ainlcizorepsorating unnatural fide bond reduction as an undesirable side re- mbuatrktheedriantcerseoafsesuicnhfreeacctieolnl-ssuurnfdaceer pshuylfshiyodlorgy-l building blocks into other biopolymers, one either reduced intentionally with a trisulfon- in or outside the cell? ated triphenylphosphine or left unreduced. action. We addressed this issue by incubating gicraolupcsowndaistionbseravrediinnsitghnisifcicaasnet(Foing. t5hBe).tiWme need not be restricted to cell surface oligo- conclude that triarylphosphines such as 4 and 5 Phosphine oxide 6, the product of the classical on March 7, 2012 Jurkat cells with triarylphosphine 4, an interme- scale of our experiments (13). Correspondingly, saccharides as hosts for these chemical han- Staudinger reaction, was prepared indepen- diate in the synthesis of 5, and quantifying the amreetaebsosleicntiastlulydieusnroefacttihvee atzoiwdoarddrudgisuAlfZidTe dles (15, 16). Azido–amino acids, for exam- dently and incubated with the cells (1 mM for appearance of free sulfhCyodruyldg rothupes opnhtohescpehllineb( aobzneidd sorteuhnyddmueicrditnheeg)s esdhciosownuedldiftii9do0ne%s ,brroeecnnoddveersirny agonflidgtha en- ot pble, ccoumldpbleeteinltyr obduioceodrthinotogoprnoateli?ns and 1 hour). Analysis by flow cytometry was per- surface with iodoacetylbiotin and FITC-avidin taiozindew, ietihthaezriidnesitsthaedmpriendisotmeriendanfotrpmatohrwmayetafobr- later targeted with phosphine probes. The formed as in Fig. 4. (B) Triarylphosphines do not (Fig. 5B). After 1 hour in the presence of 1 mM reactivity. reduce disulfide bonds at the cell surface. Jurkat cells were incubated with a 1 mM solution of , no detectable increase in free sulfhydryl In a side-by-side comparison with our pre- 4 triarylphosphine 4 or TCEP for 1 hour at room groups was observed relative to cells exposed to viously reported cell surface ketone reaction Fig. 5. Specificity of the modified Staudinger reaction. (A) Cell surface biotinylation does not temperature. The cells were centrifuged (2 min, iodoacetylbiotin alone. In a positive control ex- (2), the cell surface Staudinger process was 3000 g), washed with PBS, and diluted to a superior in several respects. Using the same proceed by classical Staudinger azide reduction periment, we incubateOd JurkaOtHcells with the followed by nonspecific acylation. Jurkat cells volume of 240 ml. Samples were combined with www.sciencemag.org trialkylphosphine TCEP (1 mM, 1 hour), a reagent concentrations, azidosugar metabolism were cultured in the presence of acetylated 3 60 ml of a solution of iodoacetylbiotin (5 mM in commercial disulfide bond reducing agent. A followed by phosphine reaction produced two- as described in Fig. 4. Cell surface azides were PBS). After incubation in the dark at room marked increase in free cell-surface sulfhydryl fold higher fluorescence than ketosugar metab- either reduced intentionally with a trisulfon- temperature for 1.5 hours, the cells were olism followed by hydrazide reaction. This may ated triphenylphosphine or left unreduced. washed with buffer, stained with FITC-avidin, groups was obsHeOrved in this caPse (Fig. 5B). OWHe and analyzed by flow cytometry. In both (A)

Phosphine oxide 6, the product of the classical on March 7, 2012 conclude that triarylphosphines such as 4 and 5 reflect either a faster reaction at the cell surface and (B), error bars represent the standard de- or more efficient metabolism of azidosugar 3 as Staudinger reaction, was prepared indepen- are essentially unOreactive toward diOsulfide dently and incubated with the cells (1 mM for viation of two replicate experiments. bonds under these conditions, rendering liga- compared with ketosugar 2 (Fig. 1). The azide 1 hour). Analysis by ﬂow cytometry was per- tion with azides the predominant pathway for has a major advantage over the ketone in that its formed as in Fig. 4. (B) Triarylphosphines do not Downloaded from reactivity. reactivity is unique in a cellular context owing reduce disulﬁde bonds at the cell surface. Jurkat In a side-by-side compTarCisoEnPwith our pre- to its abiotic nature. Ketones, by contrast, cells were incubated with a 1 mM solution of viously reported cell surface ketone reaction triarylphosphine 4 or TCEP for 1 hour at room temperature. The cells were centrifuged (2 min, (2), the cell surface Staudinger process was 3000 g), washed with PBS, and diluted to a Fig. 4. (A) Analysis of cell sur- superior in several respects. Using the same volume of 240 ml. Samples were combined with

face reaction by ﬂow cytom- www.sciencemag.org reagent concentrations, azidosugar metabolism Saxon, E.; B60emrtloozfzai,s oClu. tRio.n Socf ioednocaece 2ty0lb0i0o,t in28(57m, 2M0i0n7. etry. Jurkat cells (1.25 3 105 PBS). After incubation in the dark at room followed by phosphine reaction produced two- cells per milliliter) were cul- X temperature for 1.5 hours, the cells were fold higher fluorescence than ketosugar metab- tured in the presence or ab- washed with buffer, stained with FITC-avidin, olism followed by hydrazide reaction. This may sence (control) of acetylated 3 and analyzed by ﬂow cytometry. In both (A) (20 mM for 3 days). The cells reflect either a faster reaction at the cell surface and (B), error bars represent the standard de- or more efficient metabolism of azidosugar 3 as were washed twice with 1 ml viation of two replicate experiments. compared with ketosugar 2 (Fig. 1). The azide of buffer (0.1% fetal bovine serum in PBS, pH 7.4) and di- has a major advantage over the ketone in that its

luted to a volume of 240 ml. Downloaded from reactivity is unique in a cellular context owing Samples were added to 60 ml to its abiotic nature. Ketones, by contrast, of a solution of 5 (5 mM in PBS, pH 7.4) and incubated at room temperature for 1 hour. Fig. 4. (A) Analysis of cell sur- The cells were washed and re- face reaction by flow cytom- suspended in 100 ml of buffer, then added to 100 ml of FITC-avidin staining obtained in two replicate experiments. (B) Progress of reaction over time. etry. Jurkat cells (1.25 3 105 solution (1:250 dilution in PBS). After a 10-min incubation in the dark at Assays were performed as in (A) with 40 mM acetylated 3 and varying the cells per milliliter) were cul- 4°C, the cells were washed with 1 ml of buffer and the FITC-avidin staining duration of the reaction with compound 5. (C) pH profile of reaction. Assays tured in the presence or ab- was repeated. The cells were washed twice with buffer, then diluted to a were performed as in (A) with 40 mM acetylated 3 and varying the pH of the sence (control) of acetylated 3 volume of 300 ml for flow cytometry analysis. Similar results were buffer used during incubation with compound 5. (20 mM for 3 days). The cells were washed twice with 1 ml www.sciencemag.org SCIENCE VOL 287 17 MARCH 2000 of buffer (0.1% fetal bovine 2009 serum in PBS, pH 7.4) and diluted to a volume of 240 ml. Samples were added to 60 ml of a solution of 5 (5 mM in PBS, pH 7.4) and incubated at room temperature for 1 hour. The cells were washed and re- suspended in 100 ml of buffer, then added to 100 ml of FITC-avidin staining obtained in two replicate experiments. (B) Progress of reaction over time. solution (1:250 dilution in PBS). After a 10-min incubation in the dark at Assays were performed as in (A) with 40 mM acetylated 3 and varying the 4°C, the cells were washed with 1 ml of buffer and the FITC-avidin staining duration of the reaction with compound 5. (C) pH profile of reaction. Assays was repeated. The cells were washed twice with buffer, then diluted to a were performed as in (A) with 40 mM acetylated 3 and varying the pH of the volume of 300 ml for flow cytometry analysis. Similar results were buffer used during incubation with compound 5.

www.sciencemag.org SCIENCE VOL 287 17 MARCH 2000 2009

X Activation of a Fluorgenic Dye via the Staudinger Ligation

The lone pair of electrons on phosphorus quench the excited fluorophore Me NH O coumarin dye O N3 Me NH CO2Me N H O P O

CH3CN/H2O P N O O Ph

N O O

not fluorescent strongly fluorescent

Phosphine dye was reacted with recombinant murine dihydrofolate reductase bearing azidohomoalanine residues. N3 CO2H Unnatural amino acids incorporated during overexpression NH2 in a methionine auxotrophic E. coli strain. Fluorescence was observed over background. L - azidohomoalanine No washing or Western blotting necessary.

Lemieux, G. A.; de Graffenried, C. L.; Bertozzi, C. R. J. Am. Chem. Soc. 2003, 125, 4708-4709. X X Activation of a Fluorgenic Dye via the Staudinger Ligation

A small problem

MeO2C O CO2Me [O] P P Ph

N O O N O O

not fluorescent strongly fluorescent

Oxidation of the probe by air would provide background fluorescence.

Hangauer, M. J.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2008, 47, 2394. X X Activation of a Fluorgenic Dye via the Staudinger Ligation

Fluorescence resonance energy transfer (FRET) based probe FRET NO2 O O OH N N O O

N O N no fluorescence 2 quencher N PPh2 Me O

RN3, H2O

O O OH NO2

N O O + N N NHR HO N N PPh2 O O Me fluorescence

Hangauer, M. J.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2008, 47, 2394. X X "Traceless" Staudinger Ligation

Peptide coupling by the traceless Staudinger ligation

N Peptide 2 3 O

O Peptide 1 S PPh2 N Peptide 1 S PPh2 Peptide 2

O O H O 2 Peptide 2 Peptide 2 Peptide 1 N Peptide 1 N H PPh2

SH Ph2P SH O

Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000, 2, 1939. Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2006, 128, 8820. X X "Traceless" Staudinger Ligation

Traceless Staudinger ligation is reminiscent of native chemical ligation

O O O H2N thioesterification H2N Peptide 2 O Peptide 2 Peptide 1 SR HS Peptide 1 S

SH S-N acyl shift O Peptide 2 Peptide 1 N H O

Dawson, P. E.; Muir, T. W.; Clarklewis, I.; Kent, S. B. H. Science 1994, 266, 776. X X Staudinger Ligation

O O

Staudinger OMe N3 NHR ligation R PPh2 P(O)Ph2

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-1s-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, R. G.; Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 2686. Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature 2004, 430, 873. 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 [3 + 2] Cycloadditions

R N N N N3 R' R R'

First discovered by Arthur Michael in 1893.

In the 1950s, Rolf 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).

. CuSO4 5H2O 1 mol% sodium ascorbate 5% N Ph N N PhO Ph N N N H2O:tBuOH, RT PhO 91%

Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int. Ed. 2002, 41, 2596. Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057. X X [3 + 2] Cycloadditions R R CuLn-1 N N R' N N N R' N 1,4-disubstituted 1,2,3-triazoles

R copper(III) CuLn-2 metallacycle N N N R' + [LnCu]

Catalytic Cycle

R H

alkyne

R CuLn-2

N R CuL N R' n-1 N copper acetylide

R' N N N azide Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.; Sharpless, K. B.; Fokin, V. V. J. Am. Chem. Soc. 2005, 127, 210-216. X X Copper(I) Toxicity

. CuSO4 5H2O 1 mol% sodium ascorbate 5% N Ph N N PhO Ph N N N H2O:tBuOH, RT PhO 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. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193. Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett. 2004, 6, 2853. Link, A. J.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 11164. Speers, A. E.; Adam, B. F.; Cravatt, B. F. J. Am. Chem. Soc. 2003, 125, 4686. X X [3+2] Cycloaddition - Ring 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

N N N N N N "proceeded like an explosion"

massive bond angle deformation of the acetylene to 163o ~ 18 kcal/mol of ring strain

Wittig, G.; Krebs, A. Chem. Ber. 1961, 94, 3260. Alder, K.; Stein, G. Justus Liebigs Ann. Chem. 1931, 485, 211. X

X In Vivo Imaging of Zebrafish Embryos

O F O F

DIFO

copper-free N3 click chemistry

Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Science 2008, 320, 664. X REPORTS imaging of dynamic biological processes using To image azide-labeled glycans in vivo, or any DIFO reagents (fig. S6 and supporting on- these chemistries could be complicated by slow we incubated zebrafish embryos with either line material text). reaction kinetics or reagent toxicity. The copper- Ac4GalNAz or Ac4GalNAc from 3 to 72 hpf We then assessed global patterns of glycosyl- free click reaction of azides with difluorinated and then reacted the embryos with a DIFO– ation by incubating embryos with Ac4GalNAz cyclooctyne (DIFO) reagents (10) overcomes these AXlexa Fluor 647 conjugate (DIFO-647, fig. S2). starting at 3 hpf, followed by reaction with limitations, suggesting its potential application to Robust fluorescence was observed with virtually DIFO-647 at 12-hour intervals over a 5-day pe- in vivo imaging. no background (Fig. 2A). EvIenn a fVteriav1o-m iInmre-agrioidn. Wge obsfe rvZedeabzidrea-lafbielsedhgl yEcanms abs erarylyos We chose zebrafish as a model organism be- action with DIFO-647, the Ac4GalNAz-treated as 24 hpf (Fig. 2C and fig. S6). Starting at 60 hpf cause of their well-defined developmental pro- embryos displayed substantial fluorescence that and continuing until at least 72 hpf, we observed gram (11), emerging disease models (12), and increasedFirnsat tcimone-fdirempeendd etnhtamt aznenbera(Ffiisgh. 2gBly).cana buiorsstyinnfthlueotriecs ceenczeyimntenss iatyrein ttohelejraawnrte goifo nth, e unnatural sugar. amenability to optical imaging. The metabolic We observed no toxicity or developmental abnor- pectoral fins, and olfactory organs (Fig. 2, D and substrate peracetylated N-azidoacetylgalactosamine malities resulting from treatment with Ac GalNAz E). Thus, we focused on 60 to 72 hpf for more Zebrafish cell line ZF4 wa4s incubated with various doses of Ac4GalNAzm reacted with DIFO-488 (Ac4GalNAz) was selected on the basis of its and analyzed by flow cytometry. known incorporation into mucin-type O-linked Fig. 1. Ac GalNAz is metabolically glycoproteins in mammalian cells and mice via the 4 incorporaAtezdidient-olazbeebrleafdis hcegllly clyanssa.tes were further characterized by treatment with a DIFO-Flag peptide N-acetylgalactosamine (GalNAc) salvage pathway (A) Schematic depicting the use of (13, 14) (fig. S1). We envisioned an imaging ex- conjugate. metabolic labeling with Ac4GalNAz periment (Fig. 1A) in which zebrafish embryos and copper-free click chemistry using are incubated with Ac4GalNAz and their glycans DIFO prOobbessefrovredth ehignhon-minvoalseivceular weight species were consistent with labeled glycoproteins. are visualized by reaction with DIFO-fluorophore imaging of glycans during zebrafish conjugates (fig. S2). development. (B) Flow cytometry anal- Before performing imaging experiments, we ysis of ZFF4lacegll-scmoenttaabionliicnaglly slapbeecleides (glycoproteins like b-hexosaminidase, b-integrin, nicastrin) were known or predicted sites of mucin-type O-linked glycosylation. confirmed that the zebrafish glycan biosynthetic with Ac4GalNAz. ZF4 cells were incu- enzymes are permissive of the unnatural sugar. bated with Ac4GalNAz (0 to 100 mM, The zebrafish cell line ZF4 (15) was incubated 3 days) and subsequently reacted with with various doses of Ac4GalNAz, reacted with DIFO-488 (10 mM, 1 hour). Error bars a DIFO–Alexa Fluor 488 conjugate (DIFO-488, represent the standard deviation from on March 7, 2012 fig. S2), and analyzed by flow cytometry (Fig. 1B). three replicate samples. (C) Immuno- Robust dose-dependent metabolic labeling was ob- blot analysis of lysates from zebrafish served, similar to that of mammalian cells (13, 14). embryos at 120 hpf incubated with We further characterized the azide-labeled cell Ac4GalNAc (Ac) or Ac4GalNAz (Az), probed with horseradish peroxidase lysates by treatment with a DIFO–Flag peptide – conjugated antibody to Flag (top conjugate (10). The observed high-molecular- panel) or antibody to b-tubulin weight species were consistent with labeled (bottom panel). glycoproteins (fig. S3). We then purified the Flag- containing species (16) and identified several Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Sciewww.sciencemag.org nce 2008, 320, 664. Fig. 2. In vivo imaging of X glycoproteins (b-hexosaminidase, b-integrin 1b, glycans during zebrafish lysosome-associated membrane protein, nicastrin, development. (A and B) scavenger receptor B, and Thy1) with known Zebrafish embryos were (17–19) or predicted (20) sites of mucin-type O- metabolically labeled with linked glycosylation (fig. S4). We concluded that Ac4GalNAz (Az) or Ac4GalNAc Ac4GalNAz was metabolically incorporated into (Ac) starting at 3 hpf. (A) glycoproteins in zebrafish-derived cells. Embryos were reacted at Downloaded from We next evaluated Ac4GalNAz labeling in 72 hpf with DIFO-647 for vivo. Zebrafish embryos were incubated in media 1 hour. The right panel in- containing either Ac4GalNAz or, as a control, dicates an exposure time peracetylated GalNAc (Ac4GalNAc) from 3 to that is 20 times longer than 120 hours post-fertilization (hpf). Whole-animal that in the other two panels. lysates were then reacted with a phosphine-Flag (B) Embryos were reacted at probe (21) (fig. S2) and analyzed (Fig. 1C). 72 hpf with DIFO-647 for The labeled glycoproteins were refractory to di- 1 to 60 min. Asterisks de- gestion with peptide N-glycosidase F or chon- note autofluorescence. (C) droitinase ABC (fig. S5), which suggests that Zebrafish embryos incubated with Ac GalNAz or GalNAz is primarily incorporated into mucin- 4 Ac GalNAc (fig. S6) starting type O-linked glycoproteins. 4 at 3 hpf were reacted with DIFO-647 at 24 hpf and 1Department of Chemistry, University of California, Berkeley, subsequently at 12-hour CA 94720, USA. 2Department of Molecular and Cell Biology, intervals, viewed laterally 3 University of California, Berkeley, CA 94720, USA. Howard and ventrally (alternating Hughes Medical Institute, University of California, Berkeley, panels). (D and E) Zebrafish CA 94720, USA. 4The Molecular Foundry, Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA from (C) imaged at higher 94720, USA. magnification at 60 hpf (D) *These authors contributed equally to this work. or 72 hpf (E), viewed laterally (left panels) and ventrally (right panels). Solid arrowhead, olfactory organ; open †To whom correspondence should be addressed. E-mail: arrowhead, pectoral fin. Dotted line indicates the pharyngeal epidermis in the jaw region. Scale bars in (A) and [email protected] (C), 500 mm; in (B), (D), and (E), 200 mm. www.sciencemag.org SCIE N CE V OL 320 2 MAY 2008 665 X 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 DIFO-647 between 60 and 61 hpf and DIFO-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. R. Science 2008, 320, 664. X X In Vivo Imaging of Zebrafish Embryos

Three-dye bioimaging - DIFO-647 between 60 and 61 hpf, DIFO-488 between 62 and 62 hpf, and then DIFO-555 between 72 and 73 hpf.

jaw region olfactory region

cells kinocilia

Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Science 2008, 320, 664. X X DIFO Synthesis

1) NaH OH O OH 2) Br PCC

52% 91% O O HO

1) LHMDS, - 78o C 2) TESCl cat. LHMDS, 99%, 1:1 91%

O O OSiEt3

F F Selectfluor + 95% O O 1.0:2.2 O

Baskin, J. M.; Prescher, J. A.; Laughlin, J. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2007, 104, 16793. X X DIFO Synthesis

O OTES O 1) KHMDS, - 78 oC F F F 2) TESCl Selectfluor F

97% 74% O 4:1 desired:undesired O O

RuCl3 NaIO4 93%

OTf O F F o F F 1) KHMDS, - 78 C o F F LDA, 0 C 2) Tf2NPh 47% O 11% O O HO2C HO2C HO2C

Baskin, J. M.; Prescher, J. A.; Laughlin, J. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2007, 104, 16793. X X Cyclooctyne Analogues

F MeO F N MeO N O O O O N HOOC HOOC OH DIFO, Bertozzi Lab BARAC, Bertozzi Lab DIMAC, Bertozzi Lab k = 0.076 M-1s-1 k = 0.96 M-1s-1 more water soluble k = 0.003 M-1s-1 O

HO OBu HO Boons and Popik Labs DIBO, Boons Lab photocaged 5 steps k = 0.076 M-1s-1 nontoxic, k = 0.057 M-1s-1

Sletten, E. M.; Bertozzi, C. R. Org. Lett. 2008, 10, 3097. Ning, X. H.; Guo, J.; Wolfert, M. A.; Boons, G. J. Angew. Chem. Int. Ed. 2008, 47, 2253.

Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2009, 44, 666-676. 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 Inverse-Electron Demand Diels-Alder

R R N H R N N N -N2 N N N N N N R H R R

-1 -1 k2 = 2000 M s thioredoxin Trx SH S Trx O Trx S N O O O N O O N N

NH N N O N N HN O O HN O

O N O

R R N NH

Blackman, M. L.; Royzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008, 130, 13518-13519. X X C O M M U N I C A T I O N S Inverse-Electron Demand DieInl summary,s-Ald ae newr method for bioconjugation based on inverse- electron demand Diels - Alder chemistry has been described. The reaction proceeds with very fast rates and tolerates a broad range R R N of biological functionality. H R N N N -N2 N N Acknowledgment. This work was supported by NIH Grant N N N GM068640-01. We thank Colin ThorpeN for donating Trx and Danny R Ramadan for HPLC assistance.H We thank Ryan Mehl for insight R R into the aqueous stability of 1b. We thank Colin Thorpe and John Koh for insightful discussions. -1 -1 k2 = 2000 M s Supporting Information Available: Experiments in which HPLC was used to monitor bioconjugation reactions are described. Full experimental details and 1H,13C NMR spectra are provided. This material is available free of charge via the Internet at http://pubs.acs.org. Trx = 11700 Da References (1) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40,2004.(b)Kohn,M.;Breinbauer,R.Angew. Chem., Int. Ed. 2004, 43, 3106. (c) Chen, I.; Ting, A. Y. Curr. Opin. Biotechnol. 2005, 16, 35. Micha(d)el Antos,produ J.c M.;t = Francis,12014 M. D B.a Curr. Opin. Chem. Biol. 2006, 10, 253. (e) Hahn, M. E.; Muir, T. W. Trends Biochem. Sci. 2005, 30, 26. (f) Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2006, 128, 8820. (g) Lin, F. L.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.; Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 2686. (h) Kolakowski, R. V.; DielsShangguan,-Alder pro N.;du Sauers,ct = 1 R.22 R.;22 Williams,Da L. J. J. Am. Chem. Soc. 2006, 128, 5695. (2) Recent examples: Duckworth, B. P.; Xu, J. H.; Taton, T. A.; Guo, A.; Distefano, M. D. Bioconjugate Chem. 2006, 17, 967. (b) Gauchet, C.; Labadie, G. R.; Poulter, C. D. J. Am. Chem. Soc. 2006, 128, 9274. (c) Esser-Kahn, A. P.; Francis, M. B. Angew. Chem., Int. Ed. 2008, 47, 3751. (3) (a) Wittig, G.; Krebs, A. Chem. Ber. 1961, 94, 3260. Figure 1. (a) Rapid reactivity to form 12 was monitored by ESI-MS and (4) (a) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004, HPLC. (b,c) Crude ESI-MS data forBla11ckandman12, Min. experimentsL.; Royzen, that M.; began Fox, J. M. J. Am126. C,h 15046.em. S (b)oc. Baskin,2008, 1 J.3 M.;0, 1 Prescher,3518-13 J.51 A.;9. Laughlin, S. T.; Agard, with 15 µM Trx. N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli,X J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793. (c) Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130, 11486. Scheme 3. Synthesis of trans-Cyclooctene and Tetrazine (d) Sletten, E. M.; Bertozzi, C. R. Org. Lett. 2008, 10, 3097. (e) Ning, Derivatives X. H.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Angew. Chem., Int. Ed. 2008, 47, 2253. (5) Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew. Chem., Int. Ed. 2008, 47, 2832. (b) Song, W.; Wang, Y.; Qu, J.; Lin, Q. J. Am. Chem. Soc. 2008, 130, 9654. (6) (a) Dantas de Araujo, A.; Palomo, J. M.; Cramer, J.; Seitz, O.; Alexandrov, K.; Waldmann, H. Angew. Chem., Int. Ed. 2006, 45,296.(b)Latham- Timmons, H. A.; Wolter, A.; Roach, J. S. Nucleosides, Nucleotides Nucleic Acids 2003, 22, 1495. (c) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992. (d) Yousaf, M. N.; Mrksich, M. J. Am. Chem. Soc. 1999, 121,4286.(e)Seelig,B.;Jaschke,A. Tetrahedron Lett. 1997, 38, 7729. (7) Boger, D. L. Chem. ReV. 1986, 86, 781. (8) Thalhammer, F.; Wallfahrer, U.; Sauer, J. Tetrahedron Lett. 1990, 31,6851. (9) Hunter, D.; Nielson, D. G.; Tweakley, T., Jr. J. Chem. Soc., Perkin Trans. 1 1985, 2709, and references therein. (10) For Diels - Alder reactions of styrene and 1b in water: Wijnen, J. W.; Zavarise, S.; Engberts, J. B. F. N.; Charton, M. J. Org. Chem. 1996, 61, 2001. (11) No reaction was observed by 1H NMR after 8 h when 1a (2 10 - 2 M) available 5-amino-2-cyanopyridine (7) and 2-cyanopyridine (6) - 2 - 2 was combined with BuNH2 (2 10 M) or EtSH (2 10 M) in - 2 (Scheme 3b). The amino group of tetrazine 8 provides a handle CD3OD. When a CD3OD solution of 1b (2 10 M) was combined with - 2 for functionalization via acyl transfer (e.g., 9).15 BuNH2 (2 10 M), 50% of 1b had reacted after 4 h. In a similar experiment with EtSH (2 10 - 2 M), 50% of 1b had reacted after 10 min. To illustrate compatibility of the tetrazine ligation with proteins, When 1b (2 10 - 2 M) was allowed to stir in pure water, 20% we functionalized thioredoxin (Trx) with trans-cyclooctene deriva- decomposition of 1b was noted after 2 h. (12) The product from trans-cyclooctene and 1a aromatizes upon workup. See: tive 10. Trx is a 11.7 kDa protein that contains a single disulﬁde. Padwa, A.; Rodriguez, A.; Tohidi, M.; Fukunaga, T. J. Am. Chem. Soc. Upon reduction, the solvent exposed cysteine can be selectively 1983, 105, 933. 16 (13) Royzen, M.; Yap, G. P. A.; Fox, J. M. J. Am. Chem. Soc. 2008, 130, 3760. functionalized by maleimides. Thus, Trx (15 µM) was reduced (14) Soloducho, J.; Doskocz, J.; Cabaj, J.; Roszak, S. Tetrahedron 2003, 59, with tri(3-hydroxypropyl)phosphine (THP, 1 mM) and combined 4761. (15) Acyl transfer to 8 was most effective when aromatic acylating agents were with 10 (15 µM) in acetate buffer (pH 6). ESI mass spectral analysis employed. When aliphatic acylating agents were used, undesired reactivity (Figure 1b) indicated that most of the Trx had been consumed and (presumably via ketene formation) competes with the rate of acylation. It is likely that the rate of acyl transfer to 8 is slow because it is a very that the conjugate 11 had formed. Subsequent combination of 11 electron-deﬁcient aniline. Sugars and peptides can be appended to aromatic with 1b (30 µM) indicated that the formation of 12 was complete acylating agents via terephthaloyl linkers, e.g.: (a) Angelastro, M. R.; Baugh, L. E.; Bey, P.; Burkhart, J. P.; Chen, T.-M.; Durham, S. L.; Hare, C. M.; within 5 min. A control experiment with the cis-cyclooctene Huber, E. W.; Janusz, M. J.; Koehl, J. R.; Marquart, A. L.; Mehdi, S.; analogue of 10 gave the analogue of 11, but reaction with 1b did Peet, N. P. J. Med. Chem. 1994, 37, 4538. (b) Kanie, O.; Grotenbreg, G.; Wong, C.-H. Angew. Chem., Int. Ed. 2000, 39, 4545. (c) Czifra´k, K.; not give the analogue of 12 even after 24 h. Hadady, Z.; Docsa, T.; Gergely, P.; Schmidt, J.; Wessjohann, L.; Somsa´k, HPLC was also used to monitor the bioconjugation reactions L. Carbohydr. Res. 2006, 341, 947. that gave 11 and 12 and to demonstrate that the tetrazine ligation (16) Kallis, G. - B.; Holmgren, A. J. Biol. Chem. 1980, 255, 10261. to form 12 was fast and high yielding (see Supporting Information). JA8053805

J. AM. CHEM. SOC. 9 VOL. 130, NO. 41, 2008 13519 X Inverse-Electron Demand Diels-Alder

N N

H H OH N N CD3OD N N N HN HO H H

N N

-1 -1 k2 = 22,000 M s trans-cyclooctene ring is designed via computation (M06L/6-311+G(d,p)-optimized transition structure) Journal of the American Chemical Society COMMUNICATION

Scheme 1. Synthesis of a Highly Reactive Dienophile

Taylor, M. T.; Blackman, M. L.; Dmitrenko, O.; Fox, J. M. J. Am. Chem. Soc. 2011, 133, 9646-9649. X

trans-ring fusion, the eight-membered ring adopts a crown conformation (similar to 1a)initsminimizedconformation (Figure 3c). The barrier for the reaction between 4 and s-tetrazine, ΔG‡ =8.24kcal/mol,issimilartothatfor1a and significantly higher than that for 3.Compounds3 and 4 are diastereomers and the cyclopropyl moiety is distant from the tetrazine in each transition state. We therefore conclude that the low barrier computed for the reaction of 3 with s-tetrazine is attributable to the higher strain of the eight- membered ring. Based on these calculations, we sought to prepare compound 7 (Scheme 1). Thus, a Rh-catalyzed reaction of ethyl diazoacetate in neat21c 1,5-cyclooctadiene gave 5 in 54% yield (along with 44% Figure 3. M06L/6-311 G(d,p)-optimized transition structures for the of the separable syn-isomer).21 DIBAL reduction of 5 gave the Diels Alder reaction ofþs-tetrazine with the crown conformer of trans- 21a,22 fl À known alcohol 6. The ow-chemistry method developed in cyclooctene (a), the cis-ring fused bicyclo[6.1.0]non-4-ene 3 (b), and our laboratories was used to photoisomerize 6 to trans-isomer 7 the trans-ring fused bicyclo[6.1.0]non-4-ene 4 (c). The barrier (8.24 in 74% yield.7 kcal/mol) for the reaction of 4 with s-tetrazine is 1.29 kcal/mol higher than the analogous reaction of 3. During the completion of our studies, van Delft et al. elegantly demonstrated that cyclooctyne-based bioconjugations can be accelerated through fusion of a cyclopropane ring.21a This group dienophiles for the tetrazine trans-cyclooctene ligation have reported the synthesis of 6 and readily converted it to the been derived from cyclooct-4-enol,À 7 a derivative of which was corresponding cyclooctyne derivative for bioorthogonal labeling shown to adopt the crown conformation in a crystal structure.7 and cell imaging using 3 2 cycloaddition strategies.21a The þ 1 1 We recognized that that the eight-membered ring of bicyclo- rates of these conjugations were as high as 1.66 MÀ sÀ for [6.1.0]non-4-ene derivative 3 (Figure 3b), a trans-cyclo- nitrone cycloadditions. octene annealed to cyclopropane with a cis ring fusion, would Compound 7 combines with 2a to give product 8 in >95% be forced to adopt a strained conformation similar to that of 1b yield by 1H NMR analysis (Scheme 2). As expected,6,14,23 the (Figure 2).16 Computation was used to predict whether the initially formed 4,5-dihydropyrazine derivative 8 slowly iso- added strain in 3 would manifest in faster reactions with merizes in the presence of water to the 1,4-dihydropyrazine tetrazines. derivative 8b via the aminal intermediates 8a.24 Transition state calculations in the gas phase for the inverse The rate of the reaction between 7 and 2a was studied. As the electron demand Diels Alder reaction between s-tetrazine and reaction was too rapid for reliable rate determination by UV vis trans-cyclooctene derivativesÀ were studied by us at the M06L/ kinetics, we determined the relative rate by 1H NMR throughÀ a 6(311) G(d,p) level.17,18 The reaction between trans-cyclooctene competition experiment with trans-cyclooctene at 25 °C. NMR in the crownþ conformation (1a) and s-tetrazine proceeded with a analysis was conducted immediately upon mixing, and product barrier of ΔG‡ = 8.92 kcal/mol (Figure 3a). By comparison, the mixtures were analyzed for the formation of conjugated 4,5- reaction of s-tetrazine and cis-fused bicyclo[6.1.0]non-4-ene 3 dihydropyrazine products 8 and 9 (Figure 4). Thus, competition fi ‡ proceeded with a signi cantly lower barrier of ΔG = 6.95 kcal/ of 7 (10 equiv) and 1 (10 equiv) with 2a (6.5 mM) in CD3OD mol (Figure 3b). These barriers are consistent with those that gave a 19:1 ratio of 8:9. As the rate of the reaction between 1 and 1 1 have been calculated for other Diels Alder reactions that 2a had been previously measured to be k2 = 1140 MÀ sÀ (( proceed with fast rate constants.19 TheseÀ calculations predict 40) in MeOH, these NMR experiments show the rate of reaction 1 1 that the reaction of s-tetrazine with 3 would be 29 times faster between 7 and 2a to be k2 = 22 000 MÀ sÀ (( 2000). As than the reaction with 1a.20 inverse-demand Diels Alder reactions of tetrazines show sig- We also computed the barrier for the reaction between s-tetrazine nificant accelerationsÀ due to the hydrophobic effect,6,25 it is and trans-fused bicyclo[6.1.0]non-4-ene 4.Because4 bears a possible that rates may be even faster in aqueous solvents.26

9647 dx.doi.org/10.1021/ja201844c |J. Am. Chem. Soc. 2011, 133, 9646–9649 X Inverse-Electron Demand Diels-Alder

N N

H H CD OD OH Journal of theN AmericanN Chemical3 SocietyN COMMUNICATION N N HN HO H H Scheme 1. Synthesis of a Highly Reactive Dienophile N N

-1 -1 k2 = 22,000 M s trans-cyclooctene ring is designed via computation (M06L/6-311+G(d,p)-optimized transition structure)

Taylor, M. T.; Blackman, M. L.; Dmitrenko, O.; Fox, J. M. J. Am. Chem. Soc. 201trans1, 133-ring, 9646 fusion,-9649. the eight-membered ring adopts a crown conformation (similar to 1a)initsminimizedconformation (Figure 3c). TheX barrier for the reaction between 4 and s-tetrazine, ΔG‡ =8.24kcal/mol,issimilartothatfor1a and significantly higher than that for 3.Compounds3 and 4 are diastereomers and the cyclopropyl moiety is distant from the tetrazine in each transition state. We therefore conclude that the low barrier computed for the reaction of 3 with s-tetrazine is attributable to the higher strain of the eight- membered ring. Based on these calculations, we sought to prepare compound 7 (Scheme 1). Thus, a Rh-catalyzed reaction of ethyl diazoacetate in neat21c 1,5-cyclooctadiene gave 5 in 54% yield (along with 44% Figure 3. M06L/6-311 G(d,p)-optimized transition structures for the of the separable syn-isomer).21 DIBAL reduction of 5 gave the Diels Alder reaction ofþs-tetrazine with the crown conformer of trans- 21a,22 fl À known alcohol 6. The ow-chemistry method developed in cyclooctene (a), the cis-ring fused bicyclo[6.1.0]non-4-ene 3 (b), and our laboratories was used to photoisomerize 6 to trans-isomer 7 the trans-ring fused bicyclo[6.1.0]non-4-ene 4 (c). The barrier (8.24 in 74% yield.7 kcal/mol) for the reaction of 4 with s-tetrazine is 1.29 kcal/mol higher than the analogous reaction of 3. During the completion of our studies, van Delft et al. elegantly demonstrated that cyclooctyne-based bioconjugations can be accelerated through fusion of a cyclopropane ring.21a This group dienophiles for the tetrazine trans-cyclooctene ligation have reported the synthesis of 6 and readily converted it to the been derived from cyclooct-4-enol,À 7 a derivative of which was corresponding cyclooctyne derivative for bioorthogonal labeling shown to adopt the crown conformation in a crystal structure.7 and cell imaging using 3 2 cycloaddition strategies.21a The þ 1 1 We recognized that that the eight-membered ring of bicyclo- rates of these conjugations were as high as 1.66 MÀ sÀ for [6.1.0]non-4-ene derivative 3 (Figure 3b), a trans-cyclo- nitrone cycloadditions. octene annealed to cyclopropane with a cis ring fusion, would Compound 7 combines with 2a to give product 8 in >95% be forced to adopt a strained conformation similar to that of 1b yield by 1H NMR analysis (Scheme 2). As expected,6,14,23 the (Figure 2).16 Computation was used to predict whether the initially formed 4,5-dihydropyrazine derivative 8 slowly iso- added strain in 3 would manifest in faster reactions with merizes in the presence of water to the 1,4-dihydropyrazine tetrazines. derivative 8b via the aminal intermediates 8a.24 Transition state calculations in the gas phase for the inverse The rate of the reaction between 7 and 2a was studied. As the electron demand Diels Alder reaction between s-tetrazine and reaction was too rapid for reliable rate determination by UV vis trans-cyclooctene derivativesÀ were studied by us at the M06L/ kinetics, we determined the relative rate by 1H NMR throughÀ a 6(311) G(d,p) level.17,18 The reaction between trans-cyclooctene competition experiment with trans-cyclooctene at 25 °C. NMR in the crownþ conformation (1a) and s-tetrazine proceeded with a analysis was conducted immediately upon mixing, and product barrier of ΔG‡ = 8.92 kcal/mol (Figure 3a). By comparison, the mixtures were analyzed for the formation of conjugated 4,5- reaction of s-tetrazine and cis-fused bicyclo[6.1.0]non-4-ene 3 dihydropyrazine products 8 and 9 (Figure 4). Thus, competition fi ‡ proceeded with a signi cantly lower barrier of ΔG = 6.95 kcal/ of 7 (10 equiv) and 1 (10 equiv) with 2a (6.5 mM) in CD3OD mol (Figure 3b). These barriers are consistent with those that gave a 19:1 ratio of 8:9. As the rate of the reaction between 1 and 1 1 have been calculated for other Diels Alder reactions that 2a had been previously measured to be k2 = 1140 MÀ sÀ (( proceed with fast rate constants.19 TheseÀ calculations predict 40) in MeOH, these NMR experiments show the rate of reaction 1 1 that the reaction of s-tetrazine with 3 would be 29 times faster between 7 and 2a to be k2 = 22 000 MÀ sÀ (( 2000). As than the reaction with 1a.20 inverse-demand Diels Alder reactions of tetrazines show sig- We also computed the barrier for the reaction between s-tetrazine nificant accelerationsÀ due to the hydrophobic effect,6,25 it is and trans-fused bicyclo[6.1.0]non-4-ene 4.Because4 bears a possible that rates may be even faster in aqueous solvents.26

9647 dx.doi.org/10.1021/ja201844c |J. Am. Chem. Soc. 2011, 133, 9646–9649 Journal of the American Chemical Society COMMUNICATION

X Scheme 1. Synthesis of a Highly Reactive Dienophile Inverse-Electron Demand Diels-Alder

N N

H H OH N N CD3OD N N N HN HO H H

N N

-1 -1 k2 = 22,000 M s trans-cyclooctene ring is designed via computation (M06L/6-311+G(d,p)-optimized transition structure) trans-ring fusion, the eight-membered ring adopts a crown conformation (similar to 1a)initsminimizedconformation (Figure 3c). The barrier for the reaction between 4 and s-tetrazine, ΔG‡ =8.24kcal/mol,issimilartothatfor1a and significantly higher than that for 3.Compounds3 and 4 are diastereomers and the cyclopropyl moiety is distant from the tetrazine in each transition state. We therefore conclude that the low barrier computed for the reaction of 3 with s-tetrazine is attributable to the higher strain of the eight- membered ring. Based on these calculations, we sought to prepare compound 7 (Scheme 1). Thus, a Rh-catalyzed reaction of ethyl diazoacetate Taylor, M. T.; Blackman, M. L.; Dmitrenko, O.; Fox, J. M. J. Am. Chem. Soc. in20 neat11, 121c331,5-cyclooctadiene, 9646-9649. gave 5 in 54% yield (along with 44% 21 Figure 3. M06L/6-311 G(d,p)-optimized transition structures for the of the separable syn-isomer).X DIBAL reduction of 5 gave the Diels Alder reaction ofþs-tetrazine with the crown conformer of trans- 21a,22 fl À known alcohol 6. The ow-chemistry method developed in cyclooctene (a), the cis-ring fused bicyclo[6.1.0]non-4-ene 3 (b), and our laboratories was used to photoisomerize 6 to trans-isomer 7 the trans-ring fused bicyclo[6.1.0]non-4-ene 4 (c). The barrier (8.24 in 74% yield.7 kcal/mol) for the reaction of 4 with s-tetrazine is 1.29 kcal/mol higher than the analogous reaction of 3. During the completion of our studies, van Delft et al. elegantly demonstrated that cyclooctyne-based bioconjugations can be accelerated through fusion of a cyclopropane ring.21a This group dienophiles for the tetrazine trans-cyclooctene ligation have reported the synthesis of 6 and readily converted it to the been derived from cyclooct-4-enol,À 7 a derivative of which was corresponding cyclooctyne derivative for bioorthogonal labeling shown to adopt the crown conformation in a crystal structure.7 and cell imaging using 3 2 cycloaddition strategies.21a The þ 1 1 We recognized that that the eight-membered ring of bicyclo- rates of these conjugations were as high as 1.66 MÀ sÀ for [6.1.0]non-4-ene derivative 3 (Figure 3b), a trans-cyclo- nitrone cycloadditions. octene annealed to cyclopropane with a cis ring fusion, would Compound 7 combines with 2a to give product 8 in >95% be forced to adopt a strained conformation similar to that of 1b yield by 1H NMR analysis (Scheme 2). As expected,6,14,23 the (Figure 2).16 Computation was used to predict whether the initially formed 4,5-dihydropyrazine derivative 8 slowly iso- added strain in 3 would manifest in faster reactions with merizes in the presence of water to the 1,4-dihydropyrazine tetrazines. derivative 8b via the aminal intermediates 8a.24 Transition state calculations in the gas phase for the inverse The rate of the reaction between 7 and 2a was studied. As the electron demand Diels Alder reaction between s-tetrazine and reaction was too rapid for reliable rate determination by UV vis trans-cyclooctene derivativesÀ were studied by us at the M06L/ kinetics, we determined the relative rate by 1H NMR throughÀ a 6(311) G(d,p) level.17,18 The reaction between trans-cyclooctene competition experiment with trans-cyclooctene at 25 °C. NMR in the crownþ conformation (1a) and s-tetrazine proceeded with a analysis was conducted immediately upon mixing, and product barrier of ΔG‡ = 8.92 kcal/mol (Figure 3a). By comparison, the mixtures were analyzed for the formation of conjugated 4,5- reaction of s-tetrazine and cis-fused bicyclo[6.1.0]non-4-ene 3 dihydropyrazine products 8 and 9 (Figure 4). Thus, competition fi ‡ proceeded with a signi cantly lower barrier of ΔG = 6.95 kcal/ of 7 (10 equiv) and 1 (10 equiv) with 2a (6.5 mM) in CD3OD mol (Figure 3b). These barriers are consistent with those that gave a 19:1 ratio of 8:9. As the rate of the reaction between 1 and 1 1 have been calculated for other Diels Alder reactions that 2a had been previously measured to be k2 = 1140 MÀ sÀ (( proceed with fast rate constants.19 TheseÀ calculations predict 40) in MeOH, these NMR experiments show the rate of reaction 1 1 that the reaction of s-tetrazine with 3 would be 29 times faster between 7 and 2a to be k2 = 22 000 MÀ sÀ (( 2000). As than the reaction with 1a.20 inverse-demand Diels Alder reactions of tetrazines show sig- We also computed the barrier for the reaction between s-tetrazine nificant accelerationsÀ due to the hydrophobic effect,6,25 it is and trans-fused bicyclo[6.1.0]non-4-ene 4.Because4 bears a possible that rates may be even faster in aqueous solvents.26

9647 dx.doi.org/10.1021/ja201844c |J. Am. Chem. Soc. 2011, 133, 9646–9649 X Inverse-Electron Demand Diels-Alder

Genetically encoded norbornene directs site-specific cellular protein labelling

N N r.t. + O O N N aqueous

O R O NH N R

Orthogonal synthetase/tRNA pair used to install a norbornene-containing amino acid

Genetically coded amino acid used by some N O methanogenic archaea. H N OH H Pyrrolysyl-tRNA synthetase/tRNA is Me O NH2 CUA orthogonal to endogenous tRNAs and Pyrrolysine aminoacyl-tRNA synthetases in E. coli "The 22nd Amino Acid" and eukaryotic cells.

Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin, J. W. Nature Chem. 2012, Advanced Online Publication.X X Inverse-Electron Demand Diels-Alder

Genetically encoded norbornene directs site-specific cellular protein labelling

N N r.t. + O O N N aqueous

O R O NH N R

Orthogonal synthetase/tRNA pair used to install a norbornene-containing amino acid

H O O N NHBoc NH

NHBoc

N H2O:MeOH (95:5) HO o HO NH N N 21 C N N N N -1 -1 N k = 9 M s N N

Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin, J. W. Nature Chem. 2012, Advanced Online Publication.X X Inverse-Electron Demand Diels-Alder

Genetically encoded norbornene directs site-specific cellular protein labelling

N N r.t. + O O N N aqueous

O R O NH N R

The fluorescence of certain probes increased by a 5-10 fold increase after the cycloaddition

O H N O NH2 HN

N "turn-on" fluorophorogenic probes - tetramethylrhomdamine tetrazine can quench fluorophore via (TAMRA) energy transfer N N N N

Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin, J. W. Nature Chem. 2012, Advanced Online Publication.X X Inverse-Electron Demand Diels-Alder

Cycloaddition tested with mutated epidermal growth factor receptor (EGFR-GFP)

2 (1 mM)

eGFP TAMRA Merged + DIC

H H2N N O

COOH O

3 (1 mM) eGFP TAMRA Merged + DIC

H H2N N O Me Me COOH O Me

Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin, J. W. Nature Chem. 2012, Advanced Online Publication.X X Photochemical 1,3-Dipolar Cycloaddition

R 310 nm 10 minutes N N N N N N

fluorescent N N N 10-3 to 10-1 M-1s-1 N +

R R

Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew. Chem. Int. Ed. 2008, 47, 2832. Wang, Y.; Hu, W. J.; Song, W.; Lim, R. K. V.; Lin, Q. Org. Lett. 2008, 10, 3752. Song, W.; Wang, Y.; Lin, Q. J. Am. Chem. Soc. 2008, 130, 9654.

X Future Outlook

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. R. Angew. Chem. Int. Ed. 2009, 48, 6974-6998. X