Traceless Release of Alcohols Using Thiol-Sensitive Oxanorbornadiene Linkers

Article abstract of DOI:10.1021/acs.orglett.8b01093

A class of ester-amide oxanorbornadiene (EA-OND) molecules was developed to release alcohol cargos by succinimide formation upon addition of a thiol reagent. The resulting ring-closed adducts undergo further fragmentation by retro-Diels-Alder reaction to release a furan moiety in a manner similar to oxanorbornadiene diesters. The rates of each of these fragmentation pathways in the same medium were found to be sensitive to the steric nature of the amide substituent. Alcohol release was much faster in protic solvents than in aprotic ones, suggesting that this system may be useful for rapid response to thiols in biological environments. Accordingly, the attachment and thiol-dependent release of cholesterol was characterized as an example of the manipulation of a drug-like cargo.

Full text of DOI:10.1021/acs.orglett.8b01093

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Traceless Release of Alcohols Using Thiol-Sensitive
Oxanorbornadiene Linkers
Allison G. Aioub, Cody J. Higginson, and M. G. Finn*
School of Chemistry & Biochemistry, School of Biological Sciences, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta,
Georgia 30332, United States
S
* Supporting Information
ABSTRACT: A class of ester−amide oxanorbornadiene (EA-OND)
molecules was developed to release alcohol cargos by succinimide
formation upon addition of a thiol reagent. The resulting ring-closed
adducts undergo further fragmentation by retro-Diels−Alder reaction to
release a furan moiety in a manner similar to oxanorbornadiene diesters.
The rates of each of these fragmentation pathways in the same medium
were found to be sensitive to the steric nature of the amide substituent.
Alcohol release was much faster in protic solvents than in aprotic ones,
suggesting that this system may be useful for rapid response to thiols in
biological environments. Accordingly, the attachment and thiol-depend-
ent release of cholesterol was characterized as an example of the
manipulation of a drug-like cargo.
furan or thiomaleate−attached to the released cargo. We
elaborate here on another mode of reactivity of these
connectors that allows for the rapid release of an alcohol
moiety in a traceless fashion in response to the reaction with
thiols via intramolecular cyclization to form a succinimde.
The majority of OND-based linkages described in our
previous reports have been derived from acetylene dicarboxylic
esters. Here, we focus on analogous monoamide monoester
oxanorbornadienes (EA-ONDs, structures 4, 4′ in Figure 1).
These compounds were prepared from propynoate amides 2
(R1 = CO2R, R2 = CONHR, obtained from the corresponding
lithium propiolate and isocyanate) by a Diels−Alder reaction at
slightly higher temperatures than previously employed for the
diesters. A separable mixture of regioisomers was usually
obtained (structure 4 = 4-amide-3-ester; 4′ = 3-ester-4-amide,
referring to the position numbering shown in Figure 1).
While somewhat less electron-deficient than the diesters,
these ester−amide oxanorbornadiene reagents were found to be
reasonably electrophilic. Each gave a single dominant adduct
isomer (5) in aqueous media (slightly basic pH) or organic
solvents (with activating base) within 15−20 min at room
temperature and millimolar concentrations.3 The regioselectiv-
ity of this addition was determined primarily by electronic
factors which favor addition β to the more electron-with-
drawing ester group. When steric and electronic factors conflict,
as in thiol addition to isomer 4′, regioselectivity was preserved
but the addition rate was diminished (Supporting Information,
SI).
he controlled release of bioactive compounds from
Tmolecular modifiers or carrier scaffolds is a necessary
component of many strategies to improve the site specificity,
bioavailability, half-life, and other pharmacological features of
drugs. We have previously identified electron-deficient
oxanorbornadienes (ONDs, 1) as reactive electrophiles toward
thiols (k1 > 50 M−1 s−1 under physiological conditions),1,2
generating adducts that undergo retro-Diels−Alder cleavage
with rates that vary over an extraordinarily large range (k2 ≈ 3
× 10−6 to 0.5 min−1, Figure 1).3,4 However, the utility of this
cleavage process for drug delivery could be compromised in
some cases by the fact that it leaves residual fragments−either
Figure 1. (Top) Thiol-triggered reactivity of standard diester OND
linkers (1). (Bottom) Synthesis and thiol-triggered alcohol release
from amide-ester OND electrophiles (4).
Received: April 7, 2018
© XXXX American Chemical Society
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Rather than undergoing retro-Diels−Alder (rDA) cleavage
directly upon thiol addition as for other OND derivatives, EA-
OND adducts were found to first cyclize to the corresponding
succinimides 5 (Figure 1). This ejects an alcohol from the
adjacent ester, making this a traceless linkage if the alcohol is a
desired cargo. The imide then decomposed via rDA reaction to
the corresponding furan (3) and a thiomaleimide (7).
Following up on our original observation of this process,1 we
describe here the dependence of each of these release steps on
EA-OND structure.
The rates of succinimide formation and retro-Diels−Alder
fragmentation were determined by 1H NMR, beginning
immediately after the addition of excess β-mercaptoethanol,
as illustrated by a representative case (compound 4a) in Figure
2. The well-resolved nature of resonances for each species
allowed for concentration-vs-time measurements, which fit the
first-order kinetic behavior expected for both succinimide
formation and subsequent retro-Diels−Alder fragmentation.
Experimental details of the syntheses and kinetics are given in
the SI.
Reactions of different EA-OND compounds with β-
mercaptoethanol were followed in this manner with the results
summarized in Figure 3 for 3-amide-2-ester regioisomers. In
organic solvent (CDCl3), the structure−activity landscape
proved to be fairly flat. Thus, variations in the groups attached
to the furfurylamine nitrogen (acetyl, tosyl, mesyl, compounds
4a, 4g, 4h) gave rise to similar rates of succinimide formation
(half-lives approximately 1−3 h at ambient temperature) and
rDA fragmentation (3−8 days). Capping the acetamide group
with a methyl group (4f vs 4a) also had little effect, suggesting
that activation of the ester group by intramolecular H-bonding
was not important in accelerating the succinimide ring closure
reaction. Increasing the size of the amide group from primary
(ethyl) to secondary (isopropyl) slowed ring closure by about a
factor of 3 (4a vs 4f). The nature of the ejected alcohol had a
somewhat larger impact: formation of succinimide by release of
a secondary alcohol (cyclohexanol, compound 4b) was slower
(half-life = 7.6 h), compared to primary alcohols (methyl, ethyl,
benzyl, 3-butynyl; structures 4a, 4d, 4f−i, half-lives = 1−3 h).
Compound 4c, bearing a pendant carboxylic acid, exhibited a
slower rate of ring closure than other primary esters (half-life =
5.9 h), for reasons that are not understood at present.
Bridgehead substitution (4e) induced the expected acceleration
in the rDA rate (approximately 10-fold vs compound 4a),3,4
and also substantially faster succinimide formation (approx-
imately 14-fold).
Among the variables tested, the reaction medium had the
most dramatic effect on the rate of succinimde formation. In
methanol-d4, ring closure occurred within a few minutes upon
the addition of thiol for all primary amide EA-OND variants
tested (Figure 3, 4a−e). The same was observed in D2O for the
aqueous-soluble carboxylic acid 4c, along with a much greater
sensitivity to the nature of the amide substituent, with the
Figure 2. Representative 1H NMR spectra for calculation of
succinimide and furan product formation.
Figure 3. OND modifications and the half-life of both the succinimide and rDA transformations in CDCl3 and CD3OD (Dn = dansyl).
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isopropyl derivative cyclizing at least 50 times slower than the
ethyl derivative (Figure 4, 4l vs 4c).
Figure 4. Rates of succinimide formation and rDA cleavage in D2O
with different amide groups.
The two EA-OND regioisomers derived from the Diels−
Alder reaction of alkyne and substituted furan behaved much
differently in thiol-triggered reactions, as shown in Figure 5.
Five derivatives were studied in each case, including primary,
benzylic, and secondary esters, one substrate containing a
bridgehead methyl group, and another containing an epoxide
(providing for facile conjugate addition but no rDA
fragmentation). Thiol addition occurred primarily β to the
ester substituent, as expected from stereoelectronic consid-
erations of the Michael reaction and supported by the
1
observation of expected H NMR splitting between the C−H
moiety introduced in this process and the bridgehead C−H
unit when present (SI).
In general, adducts from isomers 4 (amide in the 4-position,
and bridgehead-H at position 5; see Figure 5) underwent much
slower succinimide-forming ring closure and rDA cleavage than
the regioisomeric adducts from 4′ (amide in the 3-position).
For example, in nonprotic solvent (CDCl3), succinimide
formation was approximately 10−100 times faster for the
more crowded (4′) adducts, and retro-Diels−Alder fragmenta-
tions were even more sensitive, with rate differences of
approximately 175-fold (4b′ adduct vs 4b adduct) to 230−
300-fold (adducts of 4a′ and 4i′ vs adducts of 4a and 4i). While
a significant number of publications have appeared in which the
retro-Diels−Alder cleavage of furan-maleimide adducts is
used,5−9 we are not aware of relevant information in the
literature on cases such as this. In analogy to previous
calculations,4 we suggest that the large rDA effect may derive
from a difference in transition state symmetry caused by the
differing substitution patterns of the thiol-succinimides, with
the faster compounds (from 4′) having adjacent quaternary
centers. As before, succinimide ring closure was very fast for
both regioisomers in protic (CD3OD) solvent, but the same
trend in rDA rates was observed, in which 4a′ cleaved faster
than 4a (60-fold) and 4b′ faster than 4b (173-fold).
Figure 5. Rates of succinimide formation and rDA cleavage comparing
different regioisomers of thiol addition. Position numbering used in
the text appears in blue on the adduct structures.
for some therapeutic applications.10 The properties of
cholesterol ester derivatives 4m and 4m′ (Figure 6) were
To demonstrate the ability of the EA-OND linkage to release
a biologically active cargo, we turned to cholesterol as an
example of a hydrophobic molecule requiring controlled release
Figure 6. Rates of succinimide formation and rDA cleavage for an EA-
OND designed to release cholesterol.
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consistent with the above studies: both the release of
cholesterol by succinimide formation (at least 180-fold) and
rDA fragmentation of the resulting succinimide (195-fold) were
much faster from 4m than from 4m′ in CDCl3. Also as before,
succinimide formation (alcohol release) was very fast even from
4m in protic solvent (CD3OD).
While the ester linkage provides a means for hydrolytic
release of alcohols or carboxylic acids in biological systems or
other environments, nonenzymatic cleavage of the ester bond is
often slow or difficult to control. We describe here a triggered
process by which an oxanorbornadiene Michael acceptor takes
on a thiol nucleophile, forming an intermediate adduct that
undergoes intramolecular ester cleavage by an amide group to
form a cyclic imide. In essence, this transduces the rapid
addition of thiol into the rapid release of alcohol. The resulting
succinimide undergoes further fragmentation by a retro-Diels−
Alder pathway, unless prevented from doing so by epoxidation
of the remaining OND double bond.
The alcohol-releasing step was much faster in protic media
than organic solvent, an observation that we find reasonable in
principle, but surprising in magnitude. In addition to a large
body of research on cyclization of aspartate residues to form
cyclic byproducts or intermediates in the context of peptide
synthesis11 and protein stability,12 a variety of reports of the
conversion of 1,2-amidoesters to succinimides have appeared
involving acceleration by base,13−16 protic acid,17,18 Lewis
acid,19 and activation of the leaving group.20,21 To our
knowledge, none that release nonactivated alcohols occurs at
rates approaching those observed here in methanol solvent.
This work expands the scope and utility of OND molecules
for the delivery of molecular cargo. In addition to the extreme
sensitivity to the nature of the solvent, the rate of succinimide
formation was most sensitive in aprotic media to the nature of
the alcohol (primary ≫ secondary) and was accelerated by the
presence of two alkyl bridgehead substituents on the
oxanorbornadiene core. However, in alcohol or aqueous
solvent, succinimde formation was rapid in all cases studied,
suggesting that this mode of release will be fast in biological
applications outside of lipophilic environments.
The retro-Diels−Alder cleavage step also provided an
unexpected result, in which succinimides derived from thiol
adducts bearing bridgehead substituents in adjacent positions
(the 4′ series in Figure 5) underwent fragmentation up to
hundreds of times faster than the less sterically crowded
regioisomer. For regioisomers 4, on the other hand, rDA
cleavage of thiol-succinimides was much slower than their
formation in any solvent tested, with bridgehead substitution
being the most important rate-controlling structural variable.
The OND family of linkages therefore continues to provide
different elements of control of fragmentation and release,
which we believe will have useful application in a number of
different fields.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mgfinn@gatech.edu.
ORCID
M. G. Finn: 0000-0001-8247-3108
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Science Foundation
(CHE 1011796) and by a research partnership between
Children’s Healthcare of Atlanta and the Georgia Institute of
Technology. C.H. gratefully acknowledges support from the
NSF Graduate Research Fellowship program.
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ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b01093.
Methods, chemical syntheses and characterization data,
and data for all kinetic experiments (PDF)
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DOI: 10.1021/acs.orglett.8b01093
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