Readily Accessible Ambiphilic Cyclopentadienes for Bioorthogonal Labeling

Article abstract of DOI:10.1021/jacs.8b02978

A new class of bioorthogonal reagents based on the cyclopentadiene scaffold is described. The diene 6,7,8,9-tetrachloro-1,4-dioxospiro[4,4]nona-6,8-diene (a tetrachlorocyclopentadiene ketal, TCK) is ambiphilic and self-orthogonal with remarkable stability. The diene reacts rapidly with a trans-cyclooctene and an endo-bicyclononyne, but slowly with dibenzoazacyclooctyne (DIBAC), allowing for tandem labeling studies with mutually orthogonal azides that react rapidly with DIBAC. TCK analogues are synthesized in three steps from inexpensive, commercially available starting materials.

Full text of DOI:10.1021/jacs.8b02978

Subscriber access provided by Universiteit Utrecht
Article
Readily Accessible Ambiphilic Cyclopentadienes for Bioorthogonal Labeling
Brian J. Levandowski, Raymond F. Gamache, Jennifer M. Murphy, and Kendall N. Houk
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b02978 • Publication Date (Web): 30 Apr 2018
Downloaded from http://pubs.acs.org on May 1, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 16
Journal of the American Chemical Society
1
2
3
4
5
6
Readily Accessible Ambiphilic Cyclopentadienes
for Bioorthogonal Labeling
7
8
9
Brian J. Levandowski†‡, Raymond F. Gamache†‡, Jennifer M. Murphy*||, and K. N. Houk*†
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†Department of Chemistry and Biochemistry, University of California, Los Angeles, California
90095
||Department of Molecular and Medical Pharmacology and Crump Institute for Molecular
Imaging, David Geffen School of Medicine, University of California, Los Angeles, California
90095
Eꢀmail: houk@chem.ucla.edu
Eꢀmail: jmmurphy@mednet.ucla.edu
Abstract: A new class of bioorthogonal reagents based on the cyclopentadiene scaffold is
described. The diene 6,7,8,9ꢀtetrachloroꢀ1,4ꢀdioxospiro[4,4]nonaꢀ6,8ꢀdiene (TCK) is ambiphilic
and selfꢀorthogonal with remarkable stability. The diene reacts rapidly with a transꢀcyclooctene
and an endoꢀbicyclononyne, but slowly with dibenzoazacyclooctyne (DIBAC), allowing for
tandem labeling studies with mutually orthogonal azides that react rapidly with DIBAC. TCK
analogs are synthesized in three steps from inexpensive, commercially available starting
materials.
1
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 16
1
2
3
4
Introduction
5
6
7
8
Bioorthogonal reactions enable the study of biomolecules in living systems for the elucidation of
biological processes.1 The strainꢀpromoted 3+2 azideꢀalkyne cycloaddition (SPAAC) developed
by Bertozzi,2 and the inverseꢀelectron demand tetrazine transꢀcyclooctene DielsꢀAlder reaction
introduced by Fox3 are bioorthogonal cycloadditions that have been utilized to study complex
interactions within biological settings. These reactions take place rapidly and selectively under
physiological conditions while avoiding reactions with nucleophiles present in cellular systems.
Research focused on improving the reactivity of the SPAAC and the inverseꢀelectron
demand DielsꢀAlder (IEDꢀDA) reactions has mostly centered around modifications of the twoꢀπꢀ
electron (dienophile or dipolarophile) component. Scheme 1 shows the reactivity of benzyl azide
(1) with some of the cyclooctynes developed for bioorthogonal applications. The introduction of
a propargylic fluoride (MOFO)4 on the cyclooctyne scaffold doubles the reactivity of the
cyclooctyne, while incorporation of a second electron deficient fluorine atom at the propargylic
position (DIFO)5 results in a 30ꢀfold increase in reactivity. The accelerated reactivity arises from
negative hyperconjugation of the σ*CꢀF stabilizing the transition state.6,7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Theoretical work by the Alabugin group guided the design of SNOꢀOCTs (sulfur,
nitrogen, and oxygen containing heterocyclic cyclooctynes) where the propargylic heteroatom is
endocyclic and antiperiplanar to the alkyne πꢀbond to maximize the stabilizing effect of the πꢀσ*
hyperconjugative interaction.8,9 Tomooka and coworkers synthesized several SNOꢀOCTS and
confirmed that the rate enhancement associated with an endocyclic propargyl heteroatom
exceeds that of exocyclic propargyl substitution.8 The hyperconjugative interaction involving the
endocyclic heteroatom in SNOꢀOCTS is stronger because it is antiperiplanar to the reactive πꢀ
bond, while the heteroatoms in MOFO and DIFO are gauche.8 While modulating the electronic
2
ACS Paragon Plus Environment

Page 3 of 16
Journal of the American Chemical Society
1
2
3
4
properties of the cycloalkyne has improved reactivity, the most reactive cyclooctynes are highly
5
6
strained
multiꢀcyclic
cyclooctynes
such
as
dibenzocyclooctyne
(DIBO),10
7
8
9
biarylazacyclooctynone (BARAC)11, dibenzoazacyclooctyne
(DIBAC)12 and endo 9ꢀ
hydroxymethylbicyclo[6.1.0]nonyne (BCN).13 Optimization of the azide cyclooctyne
cycloaddition has led to rate constants that have leveled off near 1 Mꢀ1 sꢀ1. For more rapid rate
constants, the tetrazine ligation can be used.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Cyclooctynes and second orderꢀrate constants (Mꢀ1 sꢀ1) for reactions with benzyl azide
(1). Reaction rates were measured in acetonitrile (CD3CN)a, methanol (CD3OD)b, or 3:1
CD3CN/D2Oc at ambient temperature.
Tetrazines are electronꢀdeficient, highly reactive dienes that undergo IEDꢀDA reactions
with strained dienophiles to label biomolecules of interest.14,15 The rate constants for IEDꢀDA
reactions of 3,6ꢀdiꢀ2ꢀpyridylꢀ1,2,4,5ꢀtetrazine (2) with a series of dienophiles are shown in
Scheme 2. In the IEDꢀDA reaction, the lowꢀlying LUMO of the highly electrophilic tetrazine
interacts with the HOMO of the nucleophilic and strained dienophile. Dienophiles with higher
lying HOMOs are more reactive towards tetrazines. For example, transꢀcyclooctene has a higher
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 16
1
2
3
4
5
6
7
8
9
HOMO energy than cyclooctyne and is more reactive towards tetrazines.16 An interesting
exception is in the series of cycloalkenes, from cyclopropene to cyclohexene, the reactivity
diminishes despite the increasing HOMO energies across the series.14,15,17 Recent analyses by our
group and the Bickelhaupt group showed that differences in the strength of the secondary orbital
interactions, which are especially strong with cyclopropene and weaken with increasing
cycloalkene ring size, overcome the differences in primary orbital interactions.18,19
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The tetrazineꢀtransꢀcyclooctene reactions are among the fastest bioorthogonal
cycloadditions, with rates exceeding 104 Mꢀ1 sꢀ1.3 Cyclooctynes (CO),16 norbornenes (NB),20 and
cyclopropenes (1,3ꢀCP)21,22 have been paired with tetrazines when a more stable dienophile is
required, but these reactions are considerably slower (Scheme 2). Bulky dienophiles, such as 3,3ꢀ
disubstituted cyclopropenes (3,3ꢀCP)23 and 3,3,6,6ꢀtetramethylthiacycloheptynes (TMTH),24
react poorly with tetrazines. These bulky dienophiles react with azides and allow for tandem
labeling studies studies with tetrazineꢀtransꢀcyclooctene reactions for multiꢀtarget imaging.25
The use of tetrazines in bioorthogonal chemistry is hampered by their bulkiness and vulnerability
to nucleophilic attack from biological nucleophiles.26 To address these issues the Prescher group
has developed bioorthogonal reactions with the less reactive 1,2,4ꢀtriazine scaffold.27
4
ACS Paragon Plus Environment

Page 5 of 16
Journal of the American Chemical Society
1
2
3
4
5
6
Scheme 2. Dienophiles and their secondꢀorder rate constants for reactions with 3,6ꢀdiꢀ2ꢀpyridylꢀ
1,2,4,5ꢀtetrazine (2). Reactions rates are measures in 9:1 MeOH/H2Oa or MeOHb at ambient
temperature. N/R indicates no reaction.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
New reactions are continually being developed that enable rapid, selective ligations to
study molecules in chemically complex environments.28,29 Cyclopentadiene is a classic diene that
was used by Diels and Alder in their seminal 1928 publication on the DielsꢀAlder reaction.30
Substituted cyclopentadienes have since enjoyed much success in synthesis,31,32 material
functionalization,33,34 bioconjugation,35 and chemical trapping,36,37 yet their potential in
bioorthogonal chemistry remains unexplored. We have used computational screening to probe
the reactivity of cyclopentadienes with bioorthogonal 2π cycloaddends to
design a
cyclopentadieneꢀbased bioorthogonal reaction. This method of screening reduces the toil of
tedious largeꢀscale reactivity screenings in the laboratory and vastly accelerates the discovery of
new bioorthogonal reactions by providing a short list of promising cyclopentadieneꢀbased
bioorthogonal reactions to study experimentally.
Cyclopentadiene reacts as both a diene and a dienophile in the DielsꢀAlder reaction and
readily dimerizes at room temperature.38 Highly substituted cyclopentadienes, such as
hexachlorocyclopentadiene, are reactive as dienes, but do not readily dimerize at room
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 16
1
2
3
4
5
6
7
8
9
temperature.39,40 This lack of selfꢀreactivity is referred to as “selfꢀorthogonal”. Substituents at
the 1,2 and 3,4 positions of cyclopentadiene sterically impede dimerization by clashing with the
substituents at the 5ꢀposition of the cyclopentadiene. Extensive experimental and computational
studies by the Schleyer group on the stability of 5ꢀsubstituted cyclopentadienes demonstrate that
electronegative substituents destabilize the cyclopentadiene by inducing 4π antiaromatic electron
delocalization, whereas electropositive substituents stabilize the cyclopentadiene by creating 6π
aromatic character.41,42,43 Our group expanded upon Schleyer’s work with a computational study
that predicts that the DielsꢀAlder reactivity of the cyclopentadiene is tunable through substitution
at the 5ꢀposition, and that electronegative substituents accelerate the reactivity.44,45
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Sauer's pioneering studies on the synthesis and reactivities of 1,2,4,5ꢀtetrazines with
many dienophiles have been a variety of inspiration for the design of reactions in bioorthogonal
chemistry.14,15 Sauer’s detailed reports on the synthesis and reactivities of substituted
cyclopentadienes, however, have gone relatively unrecognized.39,40 The highly reactive, selfꢀ
orthogonal, and ambiphilic properties of the tetrachlorocyclopentadiene ketal (TCK) shown in
Scheme 3 and described by Sauer, attracted our attention as a potential bioorthogonal diene.39,40
Ambiphilic molecules can react as electrophiles or nucleophiles depending on the reaction
partner. TCK, being ambiphilic, is able to react with both electronꢀdeficient dienophiles, such as
maleic anhydride, and electronꢀrich dienophiles, such as (1Z,5Z)ꢀcyclooctaꢀ1,5ꢀdiene.46 The
highly substituted nature of TCK impedes dimerization. TCK is stable at room temperature and
requires heating to 80 °C in toluene for 11 days to form 71% yield of the TCK DielsꢀAlder
dimer.37
6
ACS Paragon Plus Environment

Page 7 of 16
Journal of the American Chemical Society
1
2
3
4
Scheme 3. Tetrachlorocyclopentadiene ketal (TCK)
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Results and Discussion
We have probed computationally the bioorthogonal potential of the TCK with bioorthogonal 2π
scaffolds. The M06ꢀ2X47 functional with the 6ꢀ31G(d) basis set was used for geometry
optimizations. Single point energies were calculated with the 6ꢀ311++G(d,p) basis set and
solvation effects of water were included through use of the conductorꢀlike polarizable continuum
model (CPCM).48,49 Figure 1 shows the computed transition state structures and activation free
energies for the DielsꢀAlder reactions of TCK with the bioorthogonal cycloaddends of transꢀ
cyclooctene (TS-TCO), endoꢀbicyclononyne (TS-BCN), cyclooctyne (TS-CO), norbornene
(TS-NB), 3,3ꢀdimethylcyclopropene (TS-3,3-CP), and dibenzocyclooctyne (TS-DIBO). For
the reactions of TCK with transꢀcyclooctene, dibenzocyclooctyne, and cyclooctyne, the
computational screening reveals activation free energies of 18.1 – 20.6 kcal/mol, indicating
potential as viable partners with TCK in bioorthogonal cycloadditions. By contrast, the
activation free energies for the DielsꢀAlder reactions of TCK with norbornene, 3,3ꢀdimethyl
cyclopropene, and dibenzocyclooctyne range from 23.1 – 27.7 kcal/mol, and are too high for
bioorthogonal applications. These latter scaffolds are highly reactive with some 1,3ꢀdipoles,
providing an opportunity to develop mutually orthogonal cycloadditions.
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 16
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Transition state structures and activation free energies in kcal/mol for the DielsꢀAlder
reactions of TCK with bioorthogonal cycloaddends.
Experimental
To test our in-silico predictions and evaluate the potential of TCK as a bioorthogonal reaction
partner, the secondꢀorder rate constants of TCK with BCN and TCO cycloaddends were
measured experimentally. We chose BCN and TCO as the cycloaddends because they were
predicted to be the most reactive bioorthogonal dienophiles towards TCK from the
computational screening (Figure 1). TCK was prepared from commercially available
hexachlorocyclopentadiene according to Chang’s protocol shown in Scheme 4.46
Hexachlorocyclopentadiene 3 was treated with potassium hydroxide (KOH) and ethylene glycol
8
ACS Paragon Plus Environment

Page 9 of 16
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
to yield TCK. TCK undergoes a rapid 4+2 cycloaddition with BCN and the axial 5ꢀhydroxy
transꢀcyclooctene (TCOꢀOH) stereoisomer to give cycloadducts 4 and 5, respectively. The
reactions of TCK with BCN and TCOꢀOH give a mixture of four and two stereoisomers,
respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4. Synthesis of TCK and cycloaddition rates with TCOꢀOH and BCN.
Rate constants were measured with ultraviolet visible (UV/Vis) spectroscopy by
monitoring the disappearance of the TCK absorption peak under pseudoꢀfirst order conditions.
The experimentally observed secondꢀorder rate constants for the DielsꢀAlder reactions of TCK
with BCN and TCOꢀOH in methanol are 0.26 Mꢀ1 sꢀ1 and 0.25 Mꢀ1 sꢀ1, respectively (Figure 2).
These rate constants are comparable to previously reported SPAAC bioorthogonal labeling
approaches shown in Scheme 1.
9
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 16
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. a) Plot of rate observed vs. concentration of BCN with the slope taken as the secondꢀ
order rate constant. b) Plot of rate observed vs. concentration of TCOꢀOH with the slope taken as
the secondꢀorder rate constant.
Mutually orthogonal bioorthogonal reactions allow for dual labeling studies that monitor
multiꢀcomponent biological processes by targeting multiple biomolecules.22 Computational
screening predicts that DIBO derivatives will react poorly with TCK. To test this prediction,
TCK and DIBAC were stirred together for 20 hours at room temperature, and no cycloaddition
products were observed. 1,3ꢀdipoles such as azides react quickly with DIBAC, and poorly with
TCO derivatives as shown in Scheme 5.12,50 Scheme 5 outlines these findings and demonstrates
how tandem labeling is possible with the mutually orthogonal TCKꢀTCO and benzyl azideꢀ
DIBAC reactions.
10
ACS Paragon Plus Environment

Page 11 of 16
Journal of the American Chemical Society
1
2
3
4
5
Scheme 5. Mutual orthogonality between the TCK TCOꢀOH and benzyl azideꢀDIBAC reactions.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 6 illustrates a threeꢀstep protocol for the synthesis of a Nꢀhydroxysuccinimide
functionalized TCK to enable bioconjugation to primary amines. Ketalization of
hexachlorocyclopentadiene 3 was carried out with (±)ꢀ1,2,4ꢀbutanetriol to yield the intermediate
alcohol 6. Oxidation to the corresponding carboxylic acid 7 and subsequent coupling to Nꢀ
hydroxysuccinimide under standard conditions afforded the desired activated ester 8.
Scheme 6. Synthesis of TCK succinimidyl ester 9 for bioconjugation to primary amines.
11
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 16
1
2
3
4
5
6
7
8
9
To validate the biocompatibility of the reaction, the commercial neuropeptide, [DꢀAla2,
DꢀLeu5]ꢀEnkephalin, used to prevent neuronal damage against hypoxic or ischemic induced
brain injury was chosen for initial labeling experiments.51 Activated diene 8 was readily
conjugated to [DꢀAla2, DꢀLeu5]ꢀEnkephalin via the succinimidyl ester to afford cyclopentadieneꢀ
peptide conjugate 9. Peptideꢀconjugate 9 efficiently underwent the DielsꢀAlder cycloaddition
with Cy5ꢀTCO in ambient temperature to afford the fluorescent peptide 10, as shown in Scheme
7.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 7. Bioconjugation and fluorescence labeling of [DꢀAla2, DꢀLeu5]ꢀEnkephalin.
Many bioorthogonal reagents are sensitive to air or light, react with biological
endogenous nucleophiles, such as thiols, or are unstable as a result of strain making prolonged
labeling studies and storage difficult.11,28,52 TCK can be stored at room temperature as a white
solid with a melting point range of 64.5ꢀ65.5 °C. No decomposition or dimerization of TCK was
observed by proton NMR after stirring for 63 at 37 °C in a 1:1 CD3CN:D2O mixture with
cysteine. TCK displays is stable in aqueous media and inert to the nucleophilic thiol cysteine.
Although TCK is insoluble water, polar groups that increase solubility can be added.
12
ACS Paragon Plus Environment

Page 13 of 16
Journal of the American Chemical Society
1
2
3
4
Conclusion
5
6
7
8
With the aid of inꢀsilico computational screening, we report TCKs as a new class of
bioorthogonal reagents with reaction rates towards endoꢀBCN and TCOꢀOH that are practical
for biological labeling studies. Proof of fluorescence peptide labeling with TCK is demonstrated
using a commercial neuropeptide and the nearꢀinfrared cyanine dye, Cy5. The enduring stability
of TCK is ideal for longꢀterm applications and our computational studies suggest future tandem
labeling with azide reactions is plausible. TCK is readily synthesized from inexpensive starting
materials and stable at room temperature. The dynamic reactivity, accessibility, and stability
found in TCKs are essential for adoption as a bioorthogonal reagent.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Acknowledgements
We are grateful to the National Science Foundation (CHEꢀ1361104) and the National Institute of
Health (R01 GM109078) for financial support of this research. Computer time was provided by
the UCLA Institute for Digital Research and Education (IDRE).
Associated Content
Supporting Information
Experimental and computational details are available free of charge on the ACS Publications
website at DOI:
Author Contributions
‡B.J.L. and R.F.G. contributed equally to this work.
References
(1) Sletten, E. M.; Bertozzi, C. R. Angew. Chem. Int. Ed Engl. 2009, 48 (38), 6974–6998.
(2) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004, 126 (46), 15046–15047.
(3) Blackman, M. L.; Royzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008, 130, 13518–13519.
(4) Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R. ACS Chem. Biol. 2006, 1, 644–
648.
(5) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.;
Codelli, J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 16793–16797.
(6) Gold, B.; Shevchenko, N. E.; Bonus, N.; Dudley, G. B.; Alabugin, I. V. Chemistry. J. Org. Chem.
2012, 77, 75–89.
(7) Gold, B.; Dudley, G. B.; Alabugin, I. V. J. Am. Chem. Soc. 2013, 135, 1558–1569.
13
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 16
1
2
3
4
5
6
7
8
9
(8) Ni, R.; Mitsuda, N.; Kashiwagi, T.; Igawa, K.; Tomooka, K. Angew. Chem. Int. Ed Engl. 2015, 54,
1190–1194.
(9) Burke, E. G.; Gold, B.; Hoang, T. T.; Raines, R. T.; Schomaker, J. M. J. Am. Chem. Soc. 2017, 139,
8029–8037.
(10) Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.ꢀJ. Angew. Chem. Int. Ed Engl. 2008, 47, 2253–2255.
(11) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc. 2010, 132, 3688–3690.
(12) Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F. P. J. T.; van Hest, J. C. M.; van Delft, F.
L. Chem. Commun. 2010, 46, 97–99.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(13) Dommerholt, J.; Schmidt, S.; Temming, R.; Hendriks, L. J. A.; Rutjes, F. P. J. T.; van Hest, J. C. M.;
Lefeber, D. J.; Friedl, P.; van Delft, F. L. Angew. Chem. Int. Ed Engl. 2010, 49, 9422–9425.
(14) Thalhammer, F.; Wallfahrer, U.; Sauer, J. Tetrahedron Lett. 1990, 31, 6851–6854.
(15) Sauer, J.; Heldmann, D. K.; Hetzenegger, J.; Krauthan, J.; Sichert, H.; Schuster, J. Eur. J. Org.
Chem. 1998, 1998, 2885–2896.
(16) Chen, W.; Wang, D.; Dai, C.; Hamelberg, D.; Wang, B. Chem. Commun. 2012, 48, 1736–1738.
(17) Rademacher, P. Chem. Rev. 2003, 103, 933–975.
(18) Levandowski, B. J.; Hamlin, T. A.; Bickelhaupt, F. M.; Houk, K. N. J. Org. Chem. 2017, 82, 8668–
8675.
(19) Levandowski, B. J.; Houk, K. N. J. Am. Chem. Soc. 2016, 138, 16731–16736.
(20) Wang, D.; Chen, W.; Zheng, Y.; Dai, C.; Wang, K.; Ke, B.; Wang, B. Org. Biomol. Chem. 2014, 12,
3950–3955.
(21) Yang, J.; Šečkutė, J.; Cole, C. M.; Devaraj, N. K. Angew. Chem. Int. Ed Engl. 2012, 51, 7476–7479.
(22) Patterson, D. M.; Nazarova, L. A.; Xie, B.; Kamber, D. N.; Prescher, J. A. J. Am. Chem. Soc. 2012,
134, 18638–18643.
(23) Kamber, D. N.; Nazarova, L. A.; Liang, Y.; Lopez, S. A.; Patterson, D. M.; Shih, H.ꢀW.; Houk, K.
N.; Prescher, J. J. Am. Chem. Soc. 2013, 135, 13680–13683.
(24) Kamber, D.; Nguyen, S.; Liang, Y.; Liu, F.; Briggs, J.; Shih, H.ꢀ. W.; Houk, K. N.; Prescher, J.
Isomeric 1,2,4ꢀtriazines exhibit distinct profiles of bioorthogonal reactivity. Isomeric 1,2,4ꢀTriazines
Exhibit Distinct Profiles of Bioorthogonal Reactivity. Submitted.
(25) Karver, M. R.; Weissleder, R.; Hilderbrand, S. A. Angew. Chem. Int. Ed Engl. 2012, 51, 920–922.
(26) Karver, M. R.; Weissleder, R.; Hilderbrand, S. A. Bioconjug. Chem. 2011, 22, 2263–2270.
(27) Kamber, D. N.; Liang, Y.; Blizzard, R. J.; Liu, F.; Mehl, R. A.; Houk, K. N.; Prescher, J. A. J. Am.
Chem. Soc. 2015, 137, 8388–8391.
(28) Patterson, D. M.; Nazarova, L. A.; Prescher, J. A. ACS Chem. Biol. 2014, 9, 592–605.
(29) McKay, C. S.; Finn, M. G. Chem. Biol. 2014, 21, 1075–1101.
(30) Diels, O.; Alder, K. Justus Liebigs Ann. Chem. 1928, 460, 98–122.
(31) Paquette, L. A.; Wyvratt, M. J. Domino DielsꢀAlder Reactions. I. J. Am. Chem. Soc. 1974, 96, 4671–
4673.
(32) Horton, D.; Machinami, T.; Takagi, Y.; Bergmann, C. W.; Christoph, G. C. J. Chem. Soc. Chem.
Commun. 1983, 1164.
(33) Pang, L. S. K.; Wilson, M. A. J. Phys. Chem. 1993, 97, 6761–6763.
(34) Bian, S.; Scott, A. M.; Cao, Y.; Liang, Y.; Osuna, S.; Houk, K. N.; Braunschweig, A. B. J. Am.
Chem. Soc. 2013, 135, 9240–9243.
(35) Yousaf, M. N.; Mrksich, M. Diels−Alder J. Am. Chem. Soc. 1999, 121, 4286–4287.
(36) Binger, P.; Wedemann, P.; Goddard, R.; Brinker, U. H. J. Org. Chem. 1996, 61, 6462–6464.
(37) Wiberg, K. B.; Bartley, W. J. J. Am. Chem. Soc. 1960, 82, 6375–6380.
(38) Khambata, B. S.; Wassermann, Nature 1936, 137, 496–497.
(39) Eibler, E.; Burgemeister, T.; Höcht, P.; Prantl, B.; Roßmaier, H.; Schuhbauer, H. M.; Wiest, H.;
Sauer, J. Liebigs Ann./Recl. 1997, 1997, 2451–2469.
(40) Eibler, E.; Höcht, P.; Prantl, B.; Roßmaier, H.; Schuhbauer, H. M.; Wiest, H.; Sauer, J. Liebigs
Ann./Recl. 1997, 1997, 2471–2484.
(41) Nyulászi, L.; Schleyer, P. von R. J. Am. Chem. Soc. 1999, 121, 6872–6875.
14
ACS Paragon Plus Environment

Page 15 of 16
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
(42) von Ragué Schleyer, P.; Nyulászi, L.; Kárpáti, T. Eur. J. Org. Chem. 2003, 2003, 1923–1930.
(43) Fernández, I.; Wu, J. I.; Schleyer, P. von R. Org. Lett. 2013, 15, 2990–2993.
(44) Levandowski, B. J.; Houk, K. N. J. Org. Chem. 2015, 80, 3530–3537.
(45) Levandowski, B. J.; Zou, L.; Houk, K. N. Schleyer J. Comput. Chem. 2016, 37, 117–123.
(46) Chang, W.ꢀH.. J. Chem. Soc. 1965, 0, 2305–2312.
(47) Zhao, Y.; Truhlar, D. G. T Theor. Chem. Acc. 2008, 120, 215–241.
(48) Cossi, M.; Rega, N.; Scalmani, G.; Barone, V. J. Comput. Chem. 2003, 24, 669–681.
(49) Barone, V.; Cossi, M. J. Phys. Chem. A 1998, 102, 1995–2001.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(50) Stöckmann, H.; Neves, A. A.; Day, H. A.; Stairs, S.; Brindle, K. M.; Leeper, F. J. Chem. Commun.
2011, 47, 7203–7205.
(51) Su, T. P. J. Biomed. Sci. 2000, 7, 195–199.
(52) Murrey, H. E.; Judkins, J. C.; am Ende, C. W.; Ballard, T. E.; Fang, Y.; Riccardi, K.; Di, L.;
Guilmette, E. R.; Schwartz, J. W.; Fox, J. M.; et al. J. Am. Chem. Soc. 2015, 137, 11461–11475.
15
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 16 of 16
1
2
3
4
Table of Contents
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
16
ACS Paragon Plus Environment