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. Author manuscript; available in PMC: 2012 Jun 29.
Published in final edited form as: J Am Chem Soc. 2011 Jun 6;133(25):9646–9649. doi: 10.1021/ja201844c

Design and Synthesis of Highly Reactive Dienophiles for the Tetrazine-trans-Cyclooctene Ligation

Michael T Taylor 1, Melissa L Blackman 1, Olga Dmitrenko 1, Joseph M Fox 1,✉
PMCID: PMC3230318  NIHMSID: NIHMS301884  PMID: 21599005

Abstract

Computation was used to design a trans-cyclooctene derivative that displays enhanced reactivity in the tetrazine-trans-cycloctene ligation. The optimized derivative is a (E)-bicyclo[6.1.0]non-4-ene with a cis-ring fusion, in which the eight-membered ring is forced to adopt a highly strained ‘half-chair’ conformation. Toward 3,6-dipyridyl-s-tetrazine in MeOH at 25 °C, the strained derivative is 19 and 27 times more reactive than the parent trans-cyclooctene and 4E-cyclooct-4-enol, respectively. Toward 3,6-diphenyl-s-tetrazine in MeOH at 25 °C, the strained derivative is 160 times more reactive than the parent trans-cyclooctene.

Bioorthogonal reactions— reactions which proceed efficiently in the presence of biological functionality— have broad reaching applications that span chemistry, biology, and materials science.1 The Cu-catalyzed azide-alkyne cycloaddition— the archetypical ‘click reaction’— finds broad use and application,2 but can be limited by the cytotoxicity of Cu.3 Accordingly, a number of bioorthogonal methodologies have been advanced that proceed efficiently without the need for catalysis.3-5 In 2004, Bertozzi made a seminal advance through the development of a strain-assisted version of this transformation based upon the reaction between cyclooctyne and organic azides.4 This methodology has found significant applications as a tool for in vivo labeling,4,5 and efforts to improve reaction rates and substrate accessibility have been under continual development.4,5

Recently, our group introduced the tetrazine-trans-cyclooctene ligation (Fig 1)—a bioorthogonal reaction with unusually fast rates that is based on the cycloaddition of tetrazines and trans-cyclooctene.6 The development of this bioorthogonal reaction was enabled by a photochemical flow-reaction developed by our group for the efficient preparation of trans-cyclooctenes.7 A variety of s-tetrazine derivatives were known to react with strained alkenes,8 and we have found that 3,6-diaryl-s-tetrazines offer an excellent combination of fast reaction rates and stability for both the starting material and conjugation products.7 Thus, 3,6-Di(2-pyridyl)-s-tetrazine (2a) reacts with trans-cyclooctene (1) in 9:1 MeOH/water with k2 = 2000 M−1s.8 Amido substituted 3,6-di(2-pyridyl)-s-tetrazines (2b) are readily synthesized,6 and display excellent stability toward water and biological nucleophiles.9 Derivatives of 2b (R’ = DOTA10 or cyclic RGD peptide11) have been used by Robillard10 and our group11 for radiochemical imaging, and have been shown to participate in the tetrazine-trans-cyclooctene ligation with excellent rates. After we described the use of trans-cyclooctene for tetrazine ligations, the groups of Hilderbrand and Weissleder12 and Pipkorn and Braun13 described ligations between tetrazines and less reactive strained alkenes. However, the use of trans-cyclooctene derivatives is necessary for fast rates of reactivity. Recently, the tetrazine-trans-cyclooctene ligation has been used in applications by a number of groups,10,14 including our own.11

Figure 1.

Figure 1

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Tetrazine-trans-cyclooctene ligation

The lowest energy, ‘crown’ conformation of trans-cyclooctene15 (1a, Fig 2) bears structural analogy to the chair conformation of cyclohexane, as the methylenes in both conformations display an alternating arrangement of axial and equatorial hydrogens. Alternate conformations of trans-cyclooctene are significantly higher in energy.15a,b In a recent ab initio study, Bach calculated the ‘half chair’ conformation (1b, Fig 2) to be 5.9 kcal/mol higher in energy than the crown conformation 1a.15a Calculations at the CBS-APNO level of theory (see Supporting Information) are in close agreement, and find 1b to be 5.6 kcal/mol higher in energy than 1a.

Figure 2.

Figure 2

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Calculated relative energies (0 K) for two conformations of trans-cyclooctene at the CBS-APNO and G3 levels of theory

We speculated that the significant increase in strain energy associated with non-crown conformations of trans-cyclooctene could be used to accelerate the reactivity toward tetrazines. Previously, dienophiles for the tetrazine-trans-cyclooctene ligation have been derived from cyclooct-4-enol7 — a derivative of which was shown to adopt the crown conformation in a crystal structure.7 We recognized that that the eight-membered ring of bicyclo[6.1.0]non-4-ene derivative 3 (Figure 3b)— a trans-cyclooctene annealed to cyclopropane with a cis ring fusion— would be forced to adopt a strained conformation similar to that of 1b (Figure 2).16 Computation was used to predict whether the added strain in compound 3 would manifest in faster kinetics in reactions with tetrazines.

Figure 3.

Figure 3

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M06L/6-311+G(d,p)-optimized transition structures for the Diels-Alder reaction of s-tetrazine with the crown conformer of trans-cyclooctene (a), the cis-ring fused bicyclo[6.1.0]non-4-ene 3 (b) and the trans-ring fused bicyclo[6.1.0]non-4-ene 4 (c). The barrier (8.24 kcal/mol) for the reaction of 4 with s-tetrazine is 1.29 kcal/mol higher than the analogous reaction of 3.

Transition state calculations in the gas phase for the inverse electron demand Diels-Alder reaction between s-tetrazine and trans-cyclooctene derivatives were studied by us at the M06L/6(311)+G(d,p) level.17,18 The reaction between trans-cyclooctene in the crown conformation (1a) and s-tetrazine proceeded with a barrier of ΔG‡ = 8.92 kcal/mol (Figure 3a). By comparison, the reaction of s-tetrazine and cis-fused bicyclo[6.1.0]non-4-ene 3 proceeded with a significantly lower barrier of ΔG‡ = 6.95 kcal/mol (Figure 3b). These barriers are consistent with those that have been calculated for other Diels-Alder reactions that proceed with fast rate constants.19 These calculations predict that the reaction of s-tetrazine with 3 would be 29 times faster than the reaction with 1a.20

To provide further evidence that the low barrier for 3 was a manifestation of additional strain to the eight-membered ring, we also computed the barrier for the reaction between s-tetrazine and trans-fused bicyclo[6.1.0]non-4-ene 4. Because 4 bears a trans-ring fusion, the eight-membered ring adopts a crown conformation (similar to 1a) in its minimized conformation (Figure 3c). The barrier for the reaction between 4 and s-tetrazine, ΔG‡ = 8.24 kcal/mol, is similar to that for 1a, and significantly higher than that for 3. As compounds 3 and 4 are diastereomers and the cyclopropyl moiety is distant from the tetrazine in each transition state, the primary difference between the two compounds is the eight-membered ring conformation. 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— an analog of 3 with a functionalizable group (Scheme 1). Thus, Rh-catalyzed reaction of ethyl diazoacetate in neat21c 1,5-cyclooctadiene gave 5 in 54% yield (along with 44% of the separable syn-isomer).21 DIBAL reduction of 5 gave the known alcohol 6.21a,22 The flow-chemistry method developed in our labs was used to photoisomerize 6: active removal on AgNO3-impregnated silica gel provided the trans-isomer 7 in 74% yield.7

Scheme 1.

Scheme 1

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Synthesis of a highly reactive dienophile

During the completion of our studies, van Delft and coworkers elegantly demonstrated that cyclooctyne-based bioconjugations can be accelerated through fusion of a cyclopropane ring.21a This group reported the synthesis of compound 6, and readily converted it to the corresponding cyclooctyne derivative for bioorthogonal labeling and cell imaging using 3+2 cycloaddition strategies.21a The rates of these conjugations were as high as 1.66 M−1s−1 for nitrone cycloadditions.

Compound 7 combines with 3,6-di(2-pyridyl)-s-tetrazine to give product 8 in >95% yield by 1H NMR analysis (Scheme 2). As expected,6,14,23 the initially formed 4,5-dihydropyrazine derivative 8 slowly isomerizes in the presence of water to the 1,4-dihydropyrazine derivative 8b via the aminal intermediates 8a.24

Scheme 2.

Scheme 2

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Reaction of trans-cyclooctene 7 with 2a.

The rate of the reaction between compound 7 with 3,6-di(2-pyridyl)-s-tetrazine was studied. As the reaction was too rapid for reliable rate determination by UV-vis kinetics, we determined the relative rate by 1H NMR through a competition experiment with trans-cyclooctene at 25 °C. NMR analysis was conducted immediately upon mixing, and product mixtures were analyzed for the formation of conjugated 4,5-dihydropyrazine products 8 and 9 (Figure 4). Thus, competition of 7 (10 equiv) and 1 (10 equiv) with di(2-pyridyl)-s-tetrazine (2, 6.5 mM) in CD3OD gave an 19 : 1 ratio of 8 : 9. As the rate of the reaction between 1 and 2a had been previously measured to be k2 = 1140 M−1s−1 (+/− 40) in MeOH, these NMR experiments show the rate of reaction between 7 and 2a to be k2 = 22,000 M−1s−1 (+/− 2000). As inverse-demand Diels-Alder reactions of tetrazines show significant accelerations due to the hydrophobic effect,6,25 it is possible that rates may be even faster in aqueous solvents.26

Figure 4.

Figure 4

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Relative rates of reactions with (a) 3,6-diaryltetrazines (2a) in CD3OD at 25 °C. NMR spectra (400 MHz, CD3OD) of competition experiments are shown in the insets.

In prior studies on the tetrazine-trans-cyclooctene ligation, functionalized derivatives of 4E-cyclooct-4-enol (10) have been utilized.6,10,13,14 In a competition experiment against trans-cyclooctene (1), compound 10 reacted more slowly: compounds 9 : 11 were formed in a 1.0: 0.72 ratio. Based on these relative rates, the functionalizable alcohol 10 reacts in methanol with a rate of 820 M−1s−1 (+/− 90), and a relative rate that is 27 times slower than 7.27a

The reaction rates of 3,6-diphenyl-s-tetrazine (12) and cyclooctenes 1 and 7 were directly measured by UV-vis spectroscopy. In MeOH at 25 °C, a large rate difference was observed, as 12 reacted with 7 160 times faster than did 1. Thus, 1 reacted with a rate of 19.1 (+/− 0.2) M−1s−1, whereas 7 reacted with a rate of 3100 (+/- 50) M−1s−1. For the reaction of 1 and 12, additional rate constants were measured to be 27.2 (+/− 1.5) M−1s−1 at 35 °C, and 36.4 (+/− 1.3) M−1s−1 at 45 °C, and Eyring analysis showed ΔH‡ to be 5.41 (+/− 0.7) kcal/mol, ΔS‡ to be −33.6 (+/− 2.3) e.u., and ΔG‡ to be 15.4 (+/− 0.9) kcal/mol. Based on the relative rate data at 25 °C in MeOH, ΔG‡ for the reaction of 7 and 12 was calculated to be 12.4 (+/− 0.9) kcal/mol at 25 °C in MeOH. Computation at the M06L/6-311+G(d,p) level of theory was also used to study the Diels-Alder reactions of 12. For gas phase Diels-Alder reactions with 12 at 25 °C, the experimental ΔΔG‡ (3.0 kcal/mol) for 7 vs 1 correlated closely with the calculated ΔΔG‡ (3.34 kcal/mol) for 3 vs 1a.27b

In addition to excellent reactivity, 7 also displays excellent stability. Compound 7 shows no degradation in water or in human serum after 24 hours. Compound 7 (30 mM) also shows no decomposition upon exposure to 30 mM n-butylamine in solvent for 24 hours, or to 5 mM ethanethiol in MeOH for 12 hours.28 Compound 7 could be converted into derivatives readily. Treatment with 4-nitrophenylchloroformate21a,29 gave a carbonate which combined with N-(2-aminoethyl)maleimide to give 13 (Figure 5a). As shown in Figure 5a, the reduced form30 of the 11.7 kDa protein thioredoxin (Trx, 15 μM) could be derivatized with an excess (120 μM) of maleimide 13 to give adduct 14. Subsequent reaction with 3,6-di(2-pyridyl)-s-tetrazine (2a, 120 μM) was rapid, and the crude ESI-MS indicated that the formation of adduct 15 was completed as quickly as we were able to take a measurement (< 5 min) (Figure 5b). By contrast, Trx derivatized by a cis-cyclooctene does not react with 2a.6

Figure 5.

Figure 5

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(a) Preparation of a thioredoxin–trans-cyclooctene conjugate 14, and ligation with 2a to give adduct 15. (b) Analysis of 15 within 5 min of combination of 14 and 2a.

In summary, computation was used to design a trans-cyclooctene derivative with enhanced reactivity in the tetrazine-trans-cycloctene ligation. The optimized derivative is a (E)-bicyclo[6.1.0]non-4-ene with a cis-ring fusion, in which the eight-membered ring is forced to adopt a highly strained ‘half-chair’ conformation. The strained trans-cyclooctene derivative not only displays enhanced reactivity, but it can be easily derivatized, and bioconjugation to the protein thioredoxin has been demonstrated. The strained trans-cyclooctene displays good stability in water, serum, and in the presence of amine and thiol nucleophiles. Toward 3,6-dipyridyl-s-tetrazine in MeOH at 25 °C, the strained derivative reacts with a 2nd order rate constant of 22,000 M−1s-1, which is 19 and 27 times more reactive than the parent trans-cyclooctene and 4E-cyclooct-4-enol, respectively. Toward 3,6-diphenyl-s-tetrazine, the strained derivative reacts with a rate constant of 3,100 M−1s−1, which is 160 times faster than the parent trans-cyclooctene.

Supplementary Material

1_si_001

Acknowledgments

For financial support we thank NIH P20 RR017716 (NCCR COBRE Program). NMR spectra were obtained with instrumentation supported by NSF CRIF: MU, CHE 0840401. We thank Colin Thorpe for insightful discussions and a gift of Trx.

Footnotes

Supporting Information Available: Full experimental details, 1H and 13C-NMR spectra, kinetic and computational details are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

References

  • 1.a) Sletten EM, Bertozzi CR. Angew Chem Int Ed. 2009;48:6974. doi: 10.1002/anie.200900942. [DOI] [PMC free article] [PubMed] [Google Scholar]; b) Lim RKV, Lin Q. Chem Commun. 2010;46:1589. doi: 10.1039/b925931g. [DOI] [PMC free article] [PubMed] [Google Scholar]; b) Kalia J, Raines RT. Curr Org Chem. 2010;14:138. doi: 10.2174/138527210790069839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Click chemistry reviews: Kolb HC, Finn MG, Sharpless KB. Angew Chem Int Ed. 2001;40:2004. doi: 10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5.. Moses JE, Moorhouse AD. Chem Soc Rev. 2007;36:1249. doi: 10.1039/b613014n.. Best MD. Biochemistry. 2009;48:6571. doi: 10.1021/bi9007726.. Iha RK, Wooley KL, Nystrm AM, Burke DJ, Kade MJ, Hawker CJ. Chem Rev. 2009;109:5620. doi: 10.1021/cr900138t.. Mendal M, Tornøe CW. Chem Rev. 2008;108:2952. doi: 10.1021/cr0783479.
  • 3.Reviews of Cu-free bioconjugation chemistry: Jewett JC, Bertozzi CR. Chem Soc Rev. 2010;39:1272. doi: 10.1039/b901970g.. Becer CR, Hoogenboom R, Schubert US. Angew Chem Int Ed. 2009;48:4900. doi: 10.1002/anie.200900755.. Baskin JM, Bertozzi CR. Aldrichimica Acta. 2010;43:15.. Lutz J-F. Angew Chem Int Ed. 2008;47:2182. doi: 10.1002/anie.200705365.. Selected examples: Lim RKV, Lin Q. Chem Commun. 2010;46:7993. doi: 10.1039/c0cc02863k.. Wang Y, Song W, Hu W, Lin Q. Angew Chem Int Ed. 2009;48:5330. doi: 10.1002/anie.200901220.. Ning X, Temming RP, Dommerholt J, Guo J, Ania DB, Debets MF, Wolfert MA, Boons GJ, van Delft FL. Angew Chem Int Ed. 2010;49:3065. doi: 10.1002/anie.201000408.. For the recent development of a Cu-catalyst that functions for cell-surface labeling in alkyne/azide cycloadditions: Hong V, Steinmetz NF, Manchester M, Finn MG. Bioconjugate Chem. 2010;21:1912. doi: 10.1021/bc100272z.
  • 4.(a) Agard NJ, Prescher JA, Bertozzi CR. J Am Chem Soc. 2004;126:15046. doi: 10.1021/ja044996f. [DOI] [PubMed] [Google Scholar]; (b) Jewett JC, Sletten EM, Bertozzi CR. J Am Chem Soc. 2010;132:3688. doi: 10.1021/ja100014q. [DOI] [PMC free article] [PubMed] [Google Scholar]; (c) Chang PV, Prescher JA, Sletten EM, Baskin JM, Miller IA, Agard NJ, Lo A, Bertozzi CR. Proc Natl Acad Sci U S A. 2010;107:1821. doi: 10.1073/pnas.0911116107. [DOI] [PMC free article] [PubMed] [Google Scholar]; (d) Codelli JA, Baskin JM, Agard NJ, Bertozzi CR. J Am Chem Soc. 2008;130:11486. doi: 10.1021/ja803086r. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.(a) Poloukhtine AA, Mbua NE, Wolfert MA, Boons G-J, Popik VV. J Am Chem Soc. 2009;131:15769. doi: 10.1021/ja9054096. [DOI] [PMC free article] [PubMed] [Google Scholar]; (b) Ning X, Guo J, Wolfert MA, Boons G-J. Angew Chem Int Ed. 2008;47:2253. doi: 10.1002/anie.200705456. [DOI] [PMC free article] [PubMed] [Google Scholar]; (c) McKay CS, Moran J, Pezacki JP. Chem Commun. 2010;46:931. doi: 10.1039/b921630h. [DOI] [PubMed] [Google Scholar]
  • 6.Blackman ML, Royzen M, Fox JM. J Am Chem Soc. 2008;130:13518. doi: 10.1021/ja8053805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Royzen M, Yap GPA, Fox JM. J Am Chem Soc. 2008;130:3760. doi: 10.1021/ja8001919. [DOI] [PubMed] [Google Scholar]
  • 8.(a) Thalhammer F, Wallfahrer U, Sauer J. Tetrahedron Lett. 1990;31:6851. [Google Scholar]; (b) Boger DL. Chem Rev. 1986;86:781. [Google Scholar]
  • 9.Acetamido-substituted dipyridyltetrazines (1b) are stable for prolonged periods (>24 h) in water, and toward amines and thiols in MeOH. See ref 11.
  • 10.Rossin R, Verkerk PR, Bosch SMvd, Vulders RCM, Verel I, Lub J, Robillard MS. Angew Chem Int Ed. 2010;49:3375. doi: 10.1002/anie.200906294. [DOI] [PubMed] [Google Scholar]
  • 11.(a) Selvaraj R, Liu S, Hassink M, Huang C-w, Yap L-p, Park R, Fox JM, Li Z, Conti PS. Bioorg Med Chem Lett. 2011 doi: 10.1016/j.bmcl.2011.04.116. [DOI] [PMC free article] [PubMed] [Google Scholar]; (b) Li Z, Cai H, Hassink M, Blackman ML, Brown RCD, Conti PS, Fox JM. Chem Commun. 2010;46:8043. doi: 10.1039/c0cc03078c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Devaraj NK, Weissleder R, Hilderbrand SA. Bioconj Chem. 2008;19:2297. doi: 10.1021/bc8004446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Pipkorn R, Waldeck W, Didinger B, Koch M, Mueller G, Wiessler M, Braun K. J Peptide Sci. 2009;15:235. doi: 10.1002/psc.1108. [DOI] [PubMed] [Google Scholar]
  • 14.Devaraj NK, Upadhyay R, Haun JB, Hilderbrand SA, Weissleder R. Angew Chem Int Ed. 2009;48:7013. doi: 10.1002/anie.200903233.; Devaraj NK, Hilderbrand S, Upadhyay R, Mazitschek R, Weissleder R. Angew Chem Int Ed. 2010;19:2869. doi: 10.1002/anie.200906120.. For a subsequent study on the same F-18 probe described in ref 11b: Keliher EJ, Reiner T, Turetsky A, Hilderbrand SA, Weissleder R. ChemMedChem. 2011;6:424. doi: 10.1002/cmdc.201000426.
  • 15.(a) Bach RD. J Am Chem Soc. 2009;131:5233. doi: 10.1021/ja8094137. [DOI] [PubMed] [Google Scholar]; (b) Allinger NL, Sprague JT. J Am Chem Soc. 1972;94:5734. [Google Scholar]; (c) Leong MK, Mastryukov VS, Boggs JE. J Mol Struct. 1998;445:149. [Google Scholar]; (d) Barrows SE, Eberlein TH. J Chem Educ. 2005;82:1334. [Google Scholar]
  • 16.For prior synthetic studies on cis- and trans-fused derivatives of (E)-bicyclo[6.1.0]non-4-ene: Deyru JA, Betkouski MF. J Org Chem. 1975;40:284.
  • 17.For prior calculations on Diels-Alder reactions of tetrazines and alkynes: Cioslowski J, Sauer J, Hetzenegger J, Karcber T, Hierstetter T. J Am Chem Soc. 1993;115:1353.. For prior calculations on reactions between trans-cyclooctene and azides, see ref 15a and Schoenebeck F, Ess DH, Jones GO, Houk KN. J Am Chem Soc. 2009;131:8121. doi: 10.1021/ja9003624.. Ess DH, Houk KN. J Am Chem Soc. 2008;130:10187. doi: 10.1021/ja800009z.
  • 18.A barrier of ΔG‡ = 7.12 kcal/mol for the reaction between 1a and s-tetrazine was also calculated at the MP4SDTQ/6-31G(d) //MP2/6-311+G(d,p) level of theory. This calculation is good agreement with the barrier of 8.92 kcal/mol calculated using M06L/6-311+G(d,p) method (see Fig 3 and the Supporting Information, Table 3S). Calculations at the B3LYP/6-311+G(d,p) level overestimated the barrier for the reaction between 1a and s-tetrazine relative to MP4SDTQ/6-31G(d) //MP2/6-311+G(d,p).
  • 19.Jones GO, Guner VA, Houk KN. J Phys Chem A. 2006;110:1216. doi: 10.1021/jp052055z. [DOI] [PubMed] [Google Scholar]
  • 20.krel for 3/1a was determined according to the equation: ΔΔG‡ = −RT ln(k1/k2).
  • 21.(a) Dommerholt J, Schmidt S, Temming R, Hendriks LJA, Rutjes FPJT, van Hest JCM, Lefeber DJ, Friedl F, van Delft FL. Angew Chem Int Ed. 2010;49:9422. doi: 10.1002/anie.201003761. [DOI] [PMC free article] [PubMed] [Google Scholar]; (b) Ast W, Rheinwald G, Kerber R. Makromol Chem. 1976;177:1349. [Google Scholar]; (c) Anciaux AJ, Hubert AJ, Noels AF, Petiniot N, Teyssié P. J Org Chem. 1980;45:695. [Google Scholar]
  • 22.Dehmlow EV, Plueckebaum O. J Chem Res Miniprint. 2001;4:451. [Google Scholar]
  • 23.Aminal formation has been observed in studies on 3,4-diazanorcaradienes with alcohols and water. Steigel A, Sauer J, Kleier DA, Binsch G. J Am Chem Soc. 1972;94:2770.
  • 24.In CD3CN with 10 equiv of D2O, the conversion of 8 to 8b was 50% complete after 60 h. In 10% (202 eq.) D2O/CD3CN, the conversion to 8b was slower (12% after 84 hours) and hemiaminals 8a could be observed by 1H NMR as 8 converted to 8b over the course of 206 hours. In CD3OD, only 10% of 8 was remaining after 12 hours, with 90% aminal formation from CD3OD. In CD3OD, the formation of 8b was not observed until after 96 hours, at which point 7% of 8b was observed. See the Supporting Information for full experimental details.
  • 25.Wijnen JW, Zavarise S, Engberts JBFN, Charton M. J Org Chem. 1996;61:2001. [Google Scholar]
  • 26.We have attempted to measure the rate of the reaction between 7 and 3,6-di(2-pyridyl)-s-tetrazine in 9:1 MeOH:water, but the rate of the reaction was too fast to measure accurately by UV-vis spectroscopy. Compound 12 is too poorly soluble in aqueous solvent mixtures for direct rate measurements.
  • 27.(a) In a direct competition between 7 and 10 for 2a, the amount of 11 was below the reliable 1H NMR detection limit (<5%). (b) The barriers for the gas phase reaction between trans-cyclooctene in the crown conformation (1a) and 12 were ΔH‡ = 12.92 kcal/mol and ΔG‡ = 16.09 kcal/mol at 25 °C. The calculated barriers for the reaction of 12 and 3 were significantly lower: ΔH‡ = 9.59 kcal/mol and ΔG‡ = 12.74 kcal/mol.
  • 28.At a high concentration of ethanethiol (30 mM) in MeOH, we observed isomerization of 7 (30 mM) to 6: 0% isomerization after 2 h, 25% after 2.5 h, and 58% after 3.5 h. Because the transformation of 6 to 7 has a long induction time and does not follow second-order behavior, we suspect that the the conversion of 6 to 7 may be a radical catalyzed at high thiol concentrations. For a review of radical processes that involve low concentrations of thiol-based radicals, see: Winterbourn C, Metodiewa D. Free Radical Biology & Medicine. 1999;27:322. doi: 10.1016/s0891-5849(99)00051-9.
  • 29.(a) Ning X, Guo J, Wolfert MA, Boons G. Angew Chem Int Ed. 2008;47:2253. doi: 10.1002/anie.200705456. [DOI] [PMC free article] [PubMed] [Google Scholar]; (b) Krishnamurthy VR, Wilson JT, Cui W, Song X, Yi L, Cummings RD, Chaikof EL. Langmuir. 2010;26:7675. doi: 10.1021/la101192v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Trx (15 μM) was reduced with tri(3-hydroxypropyl)phosphine (THP, 1 mM), and combined with 12 (100 μM) in acetate buffer (pH 6). As THP also reduces maleimide functionalities, an excess of 12 was required

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Supplementary Materials

1_si_001
NIHMS301884-supplement-1_si_001.pdf (12.5MB, pdf)

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