The Influence of Substitution on Thiol-Induced Oxanorbornadiene Fragmentation

Article abstract of DOI:10.1021/acs.orglett.1c01164

Oxanorbornadienes (ONDs) undergo facile Michael addition with thiols and then fragment by a retro-Diels-Alder (rDA) reaction, a unique two-step sequence among electrophilic cleavable linkages. The rDA reaction rate was explored as a function of the furan

Full text of DOI:10.1021/acs.orglett.1c01164

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Letter
The Influence of Substitution on Thiol-Induced Oxanorbornadiene
Fragmentation
Lucrezia De Pascalis,† Mei-Kwan Yau,† Dennis Svatunek, Zhuoting Tan, Srinivas Tekkam, K. N. Houk,*
and M. G. Finn*
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ABSTRACT: Oxanorbornadienes (ONDs) undergo facile Michael addition with
thiols and then fragment by a retro-Diels−Alder (rDA) reaction, a unique two-
step sequence among electrophilic cleavable linkages. The rDA reaction rate was
explored as a function of the furan structure, with substituents at the 2- and 5-
positions found to be the most influential and the fragmentation rate to be
inversely correlated with electron-withdrawing ability. Density functional theory
calculations provided an excellent correlation with the experimentally measured
OND rDA rates.
ovalent linkages that are detachable on demand have
been used for a variety of applications including drug
these with dimethyl acetylenedicarboxylate in an exploration of
the bis(dimethyl ester) OND motif. Details of the synthesis
and characterization are provided in the Supporting
Information.
C
delivery,1,2 proteomics,3 materials development,4,5 and solid-
phase synthesis.6,7 Bond fragmentation can be induced by a
variety of different stimuli, such as heat, light, nucleophiles,
acids, bases, and enzymes.8 Oxanorbornadienes (ONDs)
derived from electron-deficient alkynes are potent electrophiles
that undergo Michael addition preferentially with thiols and
then fragment by a retro-Diels−Alder (rDA) reaction, a unique
two-step sequence among thiol-reactive linkages (Figure 1).
The results of these experiments are summarized in Figure 2.
Similar to previously reported structures,9−11 these compounds
were found to engage in rapid Michael addition with β-
mercaptoethanol to form the corresponding adducts. In each
case, this step was completed within 10−15 min at millimolar
concentrations at room temperature in organic solvent and
with equimolar activating base. We then measured the rates of
retro-Diels−Alder fragmentation to the furan and correspond-
ing thiomaleate by 1H NMR. A representative example (1a) is
shown in Figure 3, starting upon addition of excess β-
mercaptoethanol and triethylamine. Following clean Michael
addition, each adduct underwent a first-order rDA process,
chronicled by the integration of characteristic 1H NMR
resonances followed by an excellent fit to natural log vs time
plots.
Figure 1. Michael addition of a free thiol onto OND followed by
retro-Diels−Alder rearrangement to yield furan and thiomaleate.
The advantages of this system include mild cleavage
conditions, highly tunable rates of fragmentation, and ease of
synthesis. The rDA process of the thiol adduct gives furans and
thiomaleates, whereas rDA cleavage of the starting OND
occurs much more slowly in the absence of thiol.9
Modifications of the OND structure can be made which
change adduct stability toward rDA over a very wide range,
with half-lives from minutes to months.9−11 We have used the
OND system for drug cargo delivery12,13 and the generation of
degradable hydrogels.14,15
The 2,3-substituted ONDs (1a−c) were found to undergo
very slow fragmentation, with room-temperature half-lives
ranging from 16 to 34 days for compounds with methyl or
benzyl substituents at the 3-position. Limited exploration of
other disubstitution patterns (2d = 2,4; 2e = 2,3) showed
Received: April 4, 2021
Published: April 14, 2021
The rDA rates of various mono- and 2,5-disubstituted OND
have been previously reported.9,10 Here, we describe the effects
of different patterns of disubstitution on the furan component
including variations in steric and electronic properties, pairing
https://doi.org/10.1021/acs.orglett.1c01164
© 2021 American Chemical Society
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Figure 2. Structures of OND−thiol adducts explored in this study with corresponding rDA rates at room temperature in CDCl3. Experimental
error in rate constants is 10%. Compounds 3−5 are from ref 9. Compounds 6 and 7 are from ref 10. (Lower left) Linear free energy relationship
between relative rDA rate and σ+ parameter17 for 2f−2i. R = CH2CH2OH; all compounds were racemic. For compounds derived from 2,5-
disubstituted furans, thiol addition occurs to either of two positions, as indicated. rDA rates for these compounds were measured for the mixture.
shown in the inset in Figure 2, with electron donation
stabilizing the Diels−Alder transition state.
Electronic effects at the bridgehead position were further
explored with fluorinated OND derivatives 1j−1o. As
expected, fluorination gave rise to more stable thiol adducts,
with trifluoromethyl (2m) having a greater effect than
difluoromethyl (2j and 2k, differing in the substituent at the
other bridgehead position). While we did not make discrete
comparisons to nonfluorinated analogues, the aryl-substituted
compounds 2n and 2o were far more stable than other
compounds having aryl−alkyl substitution at the bridgehead
positions such as 7.10 Most striking was the great stability of
the 5-fluoro derivative 2l, which gave negligible amounts of
rDA cleavage products after one month at room temperature.
Interestingly, 5-cyclopropyl substitution provided enhanced
stability relative to methyl (2p vs 2q, 5, 6); an oxetane group
Figure 3. Addition and rDA fragmentation reactions of OND 1a as
did not have a noticeable effect (2q vs 6).
1
followed by H NMR (CDCl3, 25 °C, showing 4.5−7.4 ppm).
These structures were analyzed by density functional theory
using the SMD(chloroform)-M06-2X-D3/6-311+G(d,p) level
of theory (a detailed description of computational methods is
provided in the Supporting Information). We found an
excellent correlation between observed and calculated
energetics over 3 orders of magnitude in cycloreversion rate,
as shown in Figure 4. To gain further insight into the observed
reactivities, the parent unsubstituted system (16) was analyzed
in detail, focusing on Hirshfeld charges in both the reactant
somewhat faster cycloreversion, with half-lives of 2−4 days.
Bridgehead aromatic substitution produced a strong accel-
eration of the rDA process, with half-lives of 2−14 h for
aromatic analogues 2f−2i. While steric effects can contribute,16
electronics plays a significant role as indicated by the apparent
linear correlation of relative rate with the Hammett σ+ constant
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Letter
relatively low charges. Since stabilization of positive charges on
the furan fragment should lead to a more stabilized transition
state and therefore a lower reaction barrier, effects at the 2 or 5
positions of the furan should be stronger than at the 3 or 4
positions (Figure 5b).
Using CF3 and phenyl (Ph) as stabilizing and destabilizing
substituents, respectively, the effect of substitution in all four
possible positions was investigated (Figure 5c). As expected,
substitution at the 3 and 4 position provided only marginal
stabilization or destabilization in the free energy of activation
of the rDA reaction. However, substitution at the 2 or 5
position led to strong reductions in barrier height for phenyl
substitution and strong increases in activation energy for
trifluoromethyl.
A second important factor emerged in consideration of
unsaturated carbon substituents such as Ph. When attached to
furan positions 2 or 5, they are of course bound to a quaternary
carbon in the oxanorbornadiene reactant. In the transition
state, the conjugated system of the furan is mostly restored,
allowing for additional stabilization by conjugation with the
substituent. In contrast, such substituents in the 3 and 4
positions are already conjugated with an adjacent alkene in the
reactant, leading to much less additional stabilization in the
transition state. This difference is also reflected in the overall
calculated driving force for rDA reactions: for example, the free
energy of reaction for 9 (−6.8 kcal/mol) is significantly more
favorable than for 11 (−2.7 kcal/mol).
In conclusion, density functional theory calculations were
found to correlate well with experimental measurements of
retro-Diels−Alder reactions of OND-thiol adducts over a wide
range. These studies illuminated two electronic and orbital
concepts for the control of OND rDA rates: the electronic
nature of substituents which stabilize or destabilize positive
charge at the furan 2 and 5 positions, and the ability of
substituents to engage in π-overlap with the furan fragment,
also much more important at the 2 and 5 positions. These
insights can be used to design OND linkers for a variety of
drug delivery and materials applications.
Figure 4. Observed half-life vs calculated (DFT) activation energy for
retro-Diels−Alder reactions; letter designations refer to the
mercaptoethanol adducts of structures 2a−2q shown in Figure 2.
and the transition state (Figure 5a). In the reactant a charge of
+0.06 e was assigned to the furan part, while a much larger
charge separation with +0.26 e on the furan was calculated for
the transition state. The biggest positive charges were seen at
positions 2 and 5 of the furan, while positions 3 and 4 showed
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.1c01164.
Experimental and computational procedures; character-
ization data for new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
K. N. Houk − Department of Chemistry and Biochemistry,
University of California, Los Angeles, California 90095,
United States; orcid.org/0000-0002-8387-5261;
Email: houk@chem.ucla.edu
M. G. Finn − School of Chemistry and Biochemistry and
School of Biological Sciences, Georgia Institute of Technology,
Atlanta, Georgia 30332, United States; orcid.org/0000-
0001-8247-3108; Email: mgfinn@gatech.edu
Figure 5. (a) Analysis of Hirshfeld charges in the furan part of
reactant 16 and transition state 16TS. Charges in electrons with
hydrogens summed into heavy atoms. (b) Predicted influence on
retro Diels−Alder reactivity. (c) Effect of stabilizing and destabilizing
substituents on the free energy of activation for the retro-Diels−Alder
reaction.
Authors
Lucrezia De Pascalis − School of Chemistry and Biochemistry,
Georgia Institute of Technology, Atlanta, Georgia 30332,
United States
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Letter
(12) Higginson, C. J.; Eno, M. R.; Khan, S.; Cameron, M. D.; Finn,
M. G. Albumin-Oxanorbornadiene Conjugates Formed ex Vivo for
the Extended Circulation of Hydrophilic Cargo. ACS Chem. Biol.
2016, 11, 2320−7.
(13) Sanhueza, C. A.; Baksh, M. M.; Thuma, B.; Roy, M. D.; Dutta,
S.; Preville, C.; Chrunyk, B. A.; Beaumont, K.; Dullea, R.; Ammirati,
M.; Liu, S.; Gebhard, D.; Finley, J. E.; Salatto, C. T.; King-Ahmad, A.;
Stock, I.; Atkinson, K.; Reidich, B.; Lin, W.; Kumar, R.; Tu, M.;
Menhaji-Klotz, E.; Price, D. A.; Liras, S.; Finn, M. G.; Mascitti, V.
Efficient Liver Targeting by Polyvalent Display of a Compact Ligand
for the Asialoglycoprotein Receptor. J. Am. Chem. Soc. 2017, 139,
3528−3536.
(14) Higginson, C. J.; Kim, S. Y.; Pelaez-Fernandez, M.; Fernandez-
Nieves, A.; Finn, M. G. Modular degradable hydrogels based on thiol-
reactive oxanorbornadiene linkers. J. Am. Chem. Soc. 2015, 137,
4984−4987.
(15) Johnson, M.; Lloyd, J.; Tekkam, S.; Crooke, S. N.; Witherden,
D. A.; Havran, W. L.; Finn, M. G. Degradable Hydrogels for the
Delivery of Immune-Modulatory Proteins in the Wound Environ-
ment. ACS Appl. Bio Mater. 2020, 3, 4779.
Mei-Kwan Yau − School of Chemistry and Biochemistry,
Georgia Institute of Technology, Atlanta, Georgia 30332,
United States
Dennis Svatunek − Department of Chemistry and
Biochemistry, University of California, Los Angeles,
California 90095, United States; orcid.org/0000-0003-
1101-2376
Zhuoting Tan − Department of Chemistry and Biochemistry,
University of California, Los Angeles, California 90095,
United States
Srinivas Tekkam − School of Chemistry and Biochemistry,
Georgia Institute of Technology, Atlanta, Georgia 30332,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c01164
Author Contributions
†L.D.P. and M.-K.Y. contributed equally to this work.
(16) Fell, J. S.; Lopez, S. A.; Higginson, C. J.; Finn, M. G.; Houk, K.
N. Theoretical Analysis of the Retro-Diels-Alder Reactivity of
Oxanorbornadiene Thiol and Amine Adducts. Org. Lett. 2017, 19,
4504−4507.
(17) Hansch, C.; Leo, A.; Taft, R. W. A survey of Hammett
substituent constants and resonance and field parameters. Chem. Rev.
1991, 91, 165−195.
Funding
This work was supported by the NIH (GM101421,
AI148740), the NSF (CHE-1764328), and by the endowment
for the James A. Carlos Family Chair for Pediatric Technology
at the Georgia Institute of Technology. D.S. is grateful to the
Austrian Science Fund (FWF, J 4216-N28) and the City of
Vienna (H331849/2018) for financial support. Calculations
were performed on the Hoffman2 system provided by the
UCLA Institute for Digital Research and Education and the
Vienna Scientific Cluster.
Notes
The authors declare no competing financial interest.
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