Degradable conjugates from oxanorbornadiene reagents

Article abstract of DOI:10.1021/ja301491h

Oxanorbornadienedicarboxylate (OND) reagents were explored for purposes of binding and releasing drugs from serum albumins as representative macromolecular carriers. Being highly reactive Michael acceptors, ONDs form adducts with thiols and amines, which then undergo retro-Diels-Alder fragmentation. A study of more than 30 model adducts revealed a number of modifications that can be used to influence adduct stability. For the most reactive OND linkers, the labeling of the single available bovine serum albumin (BSA) cysteine residue was complete within minutes at a mid-micromolar concentration of reactants. While a selectivity of greater than 1000-fold for thiol over amine was observed with model amino acids, the labeling of protein amines with ONDs is fast enough to be practical, as demonstrated by the reaction with thiol-depleted BSA. The OND-amine adducts were found to be up to 15 times more stable than OND-thiol adducts, and to be sensitive to acid by virtue of a stereochemically dependent acceleration of cycloreversion. The release rate of fluorescent cargo from serum albumins was tuned by selecting the coupling partners: the available half-lives ranged from 40 min to 7 days at 37 °C. Such versatility of release profiles from protein carriers, controlled by the nature of the OND linkage, is a useful addition to the drug delivery toolbox.

Full text of DOI:10.1021/ja301491h

Article
pubs.acs.org/JACS
Degradable Conjugates from Oxanorbornadiene Reagents
Alexander A. Kislukhin, Cody J. Higginson, Vu P. Hong, and M. G. Finn*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037,
United States
S
* Supporting Information
ABSTRACT: Oxanorbornadienedicarboxylate (OND) reagents were
explored for purposes of binding and releasing drugs from serum
albumins as representative macromolecular carriers. Being highly
reactive Michael acceptors, ONDs form adducts with thiols and
amines, which then undergo retro-Diels−Alder fragmentation. A study
of more than 30 model adducts revealed a number of modifications
that can be used to influence adduct stability. For the most reactive
OND linkers, the labeling of the single available bovine serum albumin (BSA) cysteine residue was complete within minutes at a
mid-micromolar concentration of reactants. While a selectivity of greater than 1000-fold for thiol over amine was observed with
model amino acids, the labeling of protein amines with ONDs is fast enough to be practical, as demonstrated by the reaction with
thiol-depleted BSA. The OND−amine adducts were found to be up to 15 times more stable than OND−thiol adducts, and to be
sensitive to acid by virtue of a stereochemically dependent acceleration of cycloreversion. The release rate of fluorescent cargo
from serum albumins was tuned by selecting the coupling partners: the available half-lives ranged from 40 min to 7 days at 37 °C.
Such versatility of release profiles from protein carriers, controlled by the nature of the OND linkage, is a useful addition to the
drug delivery toolbox.
Scheme 1. Reaction of OND Electrophiles with BME as a
Representative Thiol
INTRODUCTION
■
Serum albumin (SA), the most abundant plasma protein in
mammals,1 is widely recognized as a long-circulating, nontoxic,
nonimmunogenic, and biodegradable carrier for the delivery of
lipophilic cargo, including endogenous metabolites and
therapeutic drugs.2 Among the most successful examples of
this approach are human SA nanoparticles carrying adsorbed
taxol3 (Abraxane), approved for the treatment of breast cancer
in 2005.
In most vertebrates, SA displays a single free cysteine residue
that accounts for 90% of extracellular thiol content1
(approximately 0.4−0.5 mM thiol in human plasma). The
abundance of SA makes it possible to directly inject thiol-
reactive electrophiles into the bloodstream, resulting in
preferential covalent binding to SA with little or no off-target
binding.4 The observation of the intended drug activity then
often requires breakdown of the albumin−drug linkage. Acid-
sensitive hydrazones and protease-cleavable peptide sequences5
have most commonly been used for this purpose. We present
here a new approach to protein-modifying linkers and prodrugs,
comprising electrophiles capable of tunable cleavage after
reaction with thiols6 and amines.7
We have recently described the conjugate addition reactivity
of 7-oxanorbornadienedicarboxylate ester (OND) reagents (1)
with thiols (Scheme 1).8 The observed second-order rate
constants of OND electrophiles are on par with those of
standard maleimides (approximately 10−100 M−1 s−1 with
glutathione, pH 7), while their aqueous stability generally
exceeds that of the maleimides.8,9 OND−thiol adducts 2 were
found to undergo retro-Diels−Alder (rDA) fragmentation,
giving furans 3 and thiomaleates 4, whereas the rDA cleavage of
the starting OND fragment is completely absent under the
same conditions.9 Thus, thiol addition constitutes a trigger for
cleavage if the OND moiety bridges the carrier and the cargo.
In our initial report, we noted a wide range of stabilities of
different versions of 2 with changes in substitution pattern. We
describe here analogous amine- and phosphine-triggered rDA
cleavage and the results of a further investigation of the factors
that contribute to cleavage rates of thiol and amine adducts,
with the purpose of creating albumin-modifying agents with a
useful range of release rates. While rDA cleavage has been
employed in the context of drug release,10 its use with
convenient electrophilic linkages has not been previously
reported.
RESULTS
■
Synthesis and Stability of OND−Thiol Adducts.
Oxanorbornadienes 1 were readily obtained from inexpensive
furans and dialkyl acetylenedicarboxylates in a single high-
yielding step. A wide range of substituents was tolerated in this
process, including sulfonamides, amides, ureas, alcohols, and
thioethers, but not thioureas. Attempted Diels−Alder reactions
Received: February 14, 2012
Published: March 28, 2012
© 2012 American Chemical Society
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of furfurylamine, as a free base or protected as various salts
(trifluoroacetate, tosylate), invariably gave complex mixtures
resulting from conjugate addition to dimethyl acetylenedicar-
boxylate. If the required acetylene precursor was inaccessible,
the desired diester could be prepared by alkylation of saponified
OND diacid salt (Supporting Information, Scheme S1).
Despite their strongly electrophilic nature, dialkyl OND esters
were found to be very stable in aqueous solutions, with the
exception of dipropargyl esters that undergo intramolecular [2
+ 2 + 2] cycloaddition.9
Adducts 2 were obtained by addition of β-mercaptoethanol
(BME) to 1 in acetonitrile using catalytic amounts of tertiary
amine base (Table 1). In all cases, the 3-exo-syn adduct was
isolated as the sole product in near-quantitative yields,
consistent with the general preference for attack on the less
hindered exo-face of [2.2.1]-bicycles. The observed coupling
between H2 and bridgehead H1 (J = 4.2 Hz) in 2a corroborated
the regiochemical assignment of substituted adducts 2b−2v, in
which the thiolic proton at C-2 resonates as a singlet. The
bridgehead methyl substituent in 2b was sufficient to direct the
nucleophile addition to the distal maleate carbon (C3), while
unsymmetric 1,4-disubstituted adducts 6b and 6c were
obtained as mixtures of C2- and C3-isomers (Table 2). This
suggests that the regioselectivity of thiol addition is governed
by the steric bulk of the bridgehead substituents.
The decomposition of oxanorbornene adducts 2 was
1
followed by H NMR in CDCl3 at room temperature. No
deviations from expected concentration-independent first-order
decay were observed even at high conversions (up to 100%),
suggesting that the reaction is irreversible. Adduct 2a, prepared
from the simplest OND, was found to have a half-life of
approximately 12 h. Introduction of one bridgehead methyl
group (2b) increased the half-life to 2.3 days. In the rest of the
dimethyl ester series, replacing 2-methylfuran with furfuryl-
amine derivatives, including sulfonamides (2c−e, 2t−v),
amides (2f−2n), urea (2o), carbamate (2p), and furfuryl
alcohol (2q), resulted in an increase in thiol adduct stability.
Fluorinated compounds 2r and 2s were very stable. Within this
series, the steric bulk of the ester group made a modest
difference (2f−h; 2j and 2k). However, the electronic
properties of the ester groups (R2) were quite influential: the
more electron-poor the ester group as indicated by the pKa of
the corresponding alcohol, the less stable the OND adduct (2i
vs 2g, 2c vs 2t−2v; see also Table S2). In three cases,
measurement of the cycloreversion rate in buffer (Figure S1)
gave similar results to those obtained in chloroform (2c, 2d,
2g).
Thiol addition to unsymmetrical 1,4-disubstituted ONDs 5
proceeded nonselectively, giving a mixture of 2-exo-syn and 3-
exo-syn-adducts 6, which were found to be relatively unstable
toward decomposition to furan 7 and thiomaleate 4-Me (Table
2). The 1,4-dimethyl-substituted adduct 6a is extremely short-
lived, decomposing by over 80% just 2 h after purification.
Sulfonamide 6b and amide 6c were somewhat more stable.
Reactions of OND with Aromatic Thiols, Phosphines,
and Tertiary Amines. Similar to maleimides,11 OND
electrophiles were found to be reactive toward other
nucleophiles. The addition of an aromatic thiol to OND 1g
produced a mixture of syn- and anti-exo addition products that
underwent rapid decomposition. Similarly, OND 1c reacted
rapidly with trimethylphosphine and tris(carboxylethyl)-
phosphine (TCEP) to give adducts that instantaneously
decomposed by rDA fragmentation (Scheme S2). In contrast,
no reaction was observed after weeks of incubation of 1c with
triphenylphosphine at high concentration. As a representative
Table 1. Half-Lives of BME Adducts 2 (Scheme 1)
a
R1
R2
half-life, days
2a
2b
2c
H
CO2Me
CO2Me
CO2Me
0.48 0.03
2.3 0.1
Me
CH2NHDansyl
23.3 0.8
b
(26.2 0.5)
4.7 0.4
2d
CH2N(Me)Dansyl
CO2Me
b
c
(4.4 0.2)
20.5 0.5
18.8 0.5
17.8 0.7
(12.6 0.4)
11.3 1.2
5.6 0.1
2e
2f
CH2NHMs
CH2NHAc
CH2NHAc
CO2Me
CO2Me
CO2Et
2g
2h
2i
CH2NHAc
CO2But
CH2NHAc
CO2CH2CCH
CO2Et
2j
CH2NHCOC6H4pNO2
CH2NHCOC6H4pNO2
CH2NHCOC6H5
CH2NHCOC6H4pOMe
19.8 0.3
20.6 0.8
16.8 0.5
16.2 0.5
14.6 0.3
7.7 0.6
2k
2l
CO2Me
CO2Me
2m
2n
2o
2p
2q
2r
2s
2t
CO2Me
CH2NHCOC6H4pNMe2 CO2Me
CH2NHCONHC6H5
CH2NHBoc
CO2Me
CO2Me
16.6 1.1
7.2 0.5
CH2OH
CO2Me
CH2NHDansyl
CH2NHDansyl
CH2NHDansyl
CH2NHDansyl
CF3, CO2Et
CF3, CF3
241 12
192 10
8.9 0.1
CO2CH2CH2CCH
2u
CO2(CH2)2O-
CH2CCH
12.1 0.2
2v
CH2N(Me)Dansyl
CO2(CH2)2O-
CH2CCH
7.0 0.1
a
b
Measured in CDCl3 by NMR. Measured in pH 7 phosphate buffer
c
by HPLC of aliquots. Measured in 9:1 buffer/DMSO by following
appearance of thiomaleate absorbance.
Table 2. Half-Lives of 1,4-Disubstituted Thiol Adducts 6
regioisomer
a
Adduct
R1
ratio
half-life
<1 h
6a
6b
6c
Me
−
2:1
CH2NHDansyl
1.9 0.1 h
3.1 0.2 h
CH2NHCOC6H4pNO2
1.3:1
a
Measured in CDCl3 by NMR.
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Scheme 2. Addition of Amines to OND Reagents and Cycloreversion of Adducts
case, OND 1k was found also to be unreactive with
triethylamine or 4-dimethylaminopyridine for at least one
week in acetonitrile, highlighting the greater stability of ONDs
to hard nucleophiles in organic solvent than to soft
nucleophiles such as thiols and phosphines.
Synthesis and Stability of OND−Amine Adducts. The
reactions of nontertiary amines with ONDs were studied in
Figure 1. Orbital and hydrogen bonding interactions (in red) that
influence the fragmentation rates of OND adducts.
closer detail to model the potential Michael addition of
proteinaceous nitrogen nucleophiles (Scheme 2, Table S1).
Adducts 8 and 9 were obtained from reactions of ONDs 1k and
5c with 1.5 equiv of 40% aq. methylamine. In the former case, a
10:1 ratio of syn and anti-isomers of structure 8 was obtained
with amine addition exclusively at C3. In the latter case, four
isomers for 9 were observed by NMR (syn and anti structures
from addition to C2 and C3), but one of these was dominant,
presumably the C3-syn adduct.
analogous diester compounds (2r, 2s vs 2c). That the attacking
heteroatom is an rDA trigger is illustrated by the observation
that furan adducts of both dimethyl maleate and dimethyl
fumarate are quite stable to rDA cycloreversion even at elevated
temperature (at least up to 65 °C).14
2. Adducts with a single bridgehead substituent (2, 8) are
100- to 300-fold more stable than the ones with 1,4-dialkyl
substitution (6, 9). The repulsion from a bridgehead methyl
group in 6 and 9 may facilitate the aforementioned hyper-
conjugative n→σ* interaction by increasing the C4−C3−X
angle. Potential transition state stabilization by the second
methyl cannot account for such a drop in stability, since the
first methyl group makes the product more stable (stability
order: 2b > 2a > 6a).
3. The presence of a stabilizing hydrogen bond between a
furfurylamine-derived NH donor and C-2 ester carbonyl
acceptor provides up to 5-fold stabilization of the thiol adducts.
Calculations suggest that this hydrogen bond is rather weak
because of suboptimal geometry (Figure S3). Nonetheless, the
rDA rates of thiol adducts 2 are correlated with the strength of
the hydrogen bond donating ability of the furfurylamine-
derived NH group at position 1 (Table 1). Within the
benzamide series (2k−2n), the stability of thiol adducts
decreases along with the decreasing acidity of para-benzamide
substituent, as reflected in the chemical shift of the amide
proton (Figure S2). In a direct test, alkylation of the NH-dansyl
group produced a 5-fold destabilization of the corresponding
dimethyl ester thiol adduct (2c vs 2d). The much less
pronounced 2-fold difference between substituted esters 2u and
2v suggests that this stabilization comes at a certain
conformational cost (structure 14, Figure 1).
4. Cycloreversion is favored by H-bonding within the maleate
fragment. Among amine adducts, the imidazole 12 is the least
stable, perhaps consistent with its ability to engage in π→σ*
donation analogous to that shown in 13, or because of its
relatively electron-deficient nature compared to methylamino
or piperidino groups. However, amine adducts are very
sensitive to protonation in a stereochemistry-dependent
Substantial differences in stability were observed for these
molecules. Anti-8 disappeared within 7 days at room temper-
ature, while syn-8 had a half-life of approximately 150 days.
Similar to the thiol adducts, 1,4-bridgehead disubstitution
induced much greater rates of retro-Diels−Alder fragmentation,
with adduct 9 showing a half-life of approximately 12 h. All
adducts 8 and 9 gave the corresponding furans and an
equilibrated12 mixture of vinylogous amides 10 (3:2 E/Z ratio
on average) in quantitative yield as judged by NMR monitoring
of the ongoing reactions. Secondary amines 4-methylpiperidine
and imidazole reacted much slower than methylamine, again
giving the syn-adducts (11 and 12) as the major products. The
syn- and anti-isomers of 11 and 12 were equally stable in both
cases (t1/2 ≈ 280 and 24 days, respectively). Interestingly,
protonation (by addition of 2 equiv of trifluoroacetic acid) of
adducts 8 and 11 made them less stable than the parent amines,
to a much greater degree for anti-8•TFA (t1/2 < 1 h) and anti-
11•TFA (t1/2 ≈ 1.9 h) than for syn-8•TFA (t1/2 ≈ 90 d) and
syn-11•TFA (t1/2 ≈ 47 d). In contrast, the anti- and syn-isomers
of 12•TFA showed a much smaller decrease in stability relative
to the free base (t1/2 ≈ 1.2 and 15 d, respectively).
Stability Trends. It is apparent that the stability of OND
adducts is governed by several stereoelectronic factors. Four
trends were identified from the above data.
1. Thiol adducts (2, 6) undergo retro-Diels−Alder fragmenta-
tion up to 15 times faster than amine adducts (8, 9). These
observations suggest transition state stabilization by n→σ*
donation, sulfur being better at this than nitrogen (structure 13,
Figure 1).13 Aromatic thiols (which degrade rapidly) are likely
to favor this interaction, whereas trifluoromethyl groups in
place of esters on the electrophile discourage it, making the
corresponding thiol adducts up to 10-fold more stable than the
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manner (Scheme 2). Thus, protonation increases the
decomposition rate of anti-adducts to a far greater degree
(20−3600-fold) than syn-adducts (1.6−6-fold). We attribute
this to the formation of an intramolecular hydrogen bond in the
anti-amine form that favors a coplanar alignment of ester and
olefinic groups (15), stimulating cycloreversion. This effect is
much less dramatic for 12•TFA, which is protonated on the sp2
imidazole nitrogen and therefore cannot engage in the
proposed activating H-bonding interaction.
Reactivity with Amino Acids. While we knew OND
reagents to be highly selective for thiol-containing substrates
such as cysteine and glutathione,8 some knowledge of their
reactivity with the large number of nonthiol nucleophilic
residues found on proteins was required. We found that the
relative and absolute reactivities of ONDs are similar to those
of maleimides. Thus, 1t reacted with a thiolate anion with an
estimated second-order rate constant of 8540 M−1 s−1 (Table
3) vs a reported value of 14200 M−1 s−1 for an N-
found to have an intrinsic reactivity with the OND electrophile
15 times greater than the N-terminal tripeptide. The substantial
amine reactivity observed for BSA with OND reagents (see
below) may therefore occur at a number of sites, depending on
such factors as lysine acidity, intrinsic nucleophilicity, and steric
accessibility.18 As with maleimides, the position(s) of attach-
ment to proteins must be assessed on a case-by-case basis.
Protein Labeling. The insights gained from the study of
model compounds can be useful for designing a degradable
material with a desired release profile. In order to demonstrate
the potential of oxanorbornadiene reagents as modular
cleavable linkers, we labeled the carrier protein bovine serum
albumin (BSA) with four fluorogenic dansyl-OND reagents
(Table 4). The substituents were chosen to provide a broad
range of half-lives for release of cargo by rDA reaction, as
inferred from the studies on small molecule adducts. BSA
contains 61 amino groups (60 lysines and N-terminus), 17
histidine imidazoles, and one free cysteine residue. Although
Cys-34 of BSA is not engaged in disulfide bonding within the
protein, it is often blocked as a mixed disulfide with small thiols
such as cysteine and can undergo aerobic oxidation to the
corresponding sulfenic acid and other products.22 Thus, the
sulfhydryl content in commercial albumin samples usually
varies from 0 to 70 mol % relative to protein concentration. In
our case, a BSA sample with a thiol content of 11% (as
determined by a modified Ellman assay23) was reduced by a
brief treatment with dithiothreitol (DTT)24 and purified by
size-exclusion filtration. The resulting reduced BSA (rBSA)
exhibited a thiol content of up to 95%.
The reaction profiles of BSA and rBSA with OND reagents
were biphasic, showing an initial burst of fluorescence followed
by a much slower nearly linear rise (Figure 2, Table 4). The
magnitude of the initial reaction was found to correspond to
the protein thiol content measured by the Ellman assay and
thus is assigned to the addition of protein thiol (Cys34) to
OND. The second phase of the BSA−OND reaction is
proposed to be due to a much slower addition to amine
nucleophiles.16 Initial second-order rate constants of OND−
amine reactions were obtained from the fluorogenic reactions
with unreduced BSA (Figure 2, dotted lines; Table 4). The
values for kamines represent the sum of initial rate constants of all
BSA nitrogen nucleophiles with each OND electrophile. For
simplicity, the starting amine concentration was set equal to the
protein concentration in the extraction of rate constants from
the data.
Table 3. Reactivity of OND 1t with Model Peptidic
Nucleophiles
Nucleophile
kobs
,
kNu
,
a
b
c
(pKa)
M−1 s−1
M−1s−1
8540
krel (Nu)
N-acetylcysteine
64.3 5.1
143000
(9.52)15
H-GlyGlyGly-OH
(10.7 1.2)
0.0596
0.918
1
(8.06)19
× 10−3
6-aminocaproate
(10.80)20
(0.36 0.03) ×
15.4
0.0095
10−3
N-benzoylhistidine
(0.38 0.04) ×
0.00057
(7.08)21
10−3
a
Observed second-order rate constant in 0.2 M sodium phosphate, pH
b
7.4, 25 °C. Reactivity of the nucleophilic form of an amino acid: kNu
=
kobs•([NuH+]/[Nu]), where Nu is the deprotonated (active) form of
the nucleophile, the concentration of which was calculated from its
c
reported pKa. Relative rate constants calculated from the preceding
column.
alkylmaleimide.15 The Michael reaction of tri(glycine), a
model for N-terminal protein amine,16 with 1t (0.2 M
phosphate, pH 7.4, 25 °C) proceeded with k2 = 0.011
0.002 M−1 s−1, again similar to the reported reaction of N-
ethylmaleimide with N-terminal valine under the same
conditions (kobs = 0.006 M−1 s−1).17 N-Benzoylhistidine and
6-aminocaproate (model compounds for histidine and lysine)
reacted much slower (Table 3). However, when corrected for
the pre-equilibrium protonation state of each nucleophile
(giving kNu and krel(Nu), Table 3), the model lysine amine was
The observed rate constant of reaction of 1t with BSA was
measured as a function of pH from 6.0 to 8.0. Fitting those
Table 4. Reactivity of BSA with OND Electrophiles and Half-Lives of the Resulting Adducts
BSA-thiol−OND
BSA-amine−OND
kthiol
kamines
kinetic
selectivity
adducts
adducts
per rBSA
adduct half-life
adduct half-life
a
b
c
d
e
f
f
OND
(M−1s−1
)
(M−1 s−1
)
per BSA
(days)
(days)
1t
44.9 1.4
27.7 1.9
15.4 0.6
10.9 0.5
0.926 0.089
0.823 0.087
0.403 0.048
0.146 0.026
48
34
38
5
4
5
0.89 0.11
0.79 0.11
0.47 0.11
0.22 0.12
1.57 0.23
1.42 0.41
1.18 0.20
0.55 0.08
0.48 0.06
0.16 0.01
0.03 0.01
6.7 0.3
6.9 0.4
4.5 0.3
0.41 0.04
1u
1v
5b
g
75 14
0.48 0.05
a
b
Observed initial second-order rate constant for reaction of rBSA thiol with OND reagents. Observed initial rate constant for secondary reaction
c
d
with amines on BSA; starting amine concentration was set equal to protein concentration. Rate ratio kthiol/kamine
. At 2 h with unreduced BSA.
Calculated from the ratio of [dansyl] (determined by absorbance at 332 nm) and [protein] (determined by Bradford assay), normalized to initial
e
f
[OND]. At 2 h with reduced BSA (thiol content of 65.0 1.0 μM by Ellman’s assay). Calculated as for (d). At 37 °C, calculated from first-order
rate constants for the rDA fragmentation of rBSA-OND adducts by extraction of liberated dansyl-furan at various time points and monitoring
remaining aqueous fluorescence. This value is lower than expected because of simultaneous rDA cleavage during the analysis procedure.
g
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Figure 2. Labeling of BSA and rBSA (70 μM) with the indicated fluorogenic OND reagents (190 μM); pH 7.0, 23 °C. Concentration of active thiol
of rBSA for reactions with 1t, 1u, 5b = 65 μM; for reaction with 1v = 40 μM.
rates to the calculated protonation state of a single amine
nucleophile gave an apparent pKa of 7.9 for this reaction site, a
value well within the possible range of lysine side chain pKa
(Figures S4 and S5).18 The precise locations of OND-amine
labeling on BSA were not determined.
Release from BSA. Tests of retro-Diels−Alder release from
BSA were performed as shown in Scheme 3. The protein was
Scheme 3. BSA Labeling and the Retro-Diels−Alder
Breakdown of the Labeled Protein
Figure 3. Kinetic profiles for retro-Diels−Alder fragmentation of
rBSA-adducts of the indicated ONDs. Straight lines mark the linear
approximations for the two first-order processes (thiol and amine
adduct release) that are assumed to contribute to the overall reaction.
degrading protonated form and slow-degrading free base form
are accessible at physiological conditions.
The results, summarized in the last two columns of Table 4,
show that (a) the bridgehead disubstituted case 5b provided
the shortest-lived adducts, (b) sulfonamide alkylation led to a
labeled with a moderate excess of fluorogenic OND reagents
decrease in thiol adduct half-life (1u vs 1v), and (c) BSA-amine
under conditions designed to promote the complete alkylation
adducts were generally found to be more stable than BSA-thiol
of available thiol and a modest level of reaction with protein
adducts. Each of these findings correlated well with the
amino groups. After removal of excess OND reagent by organic
extraction, labeling yields were estimated by measuring the dye
absorbance of aliquots of the aqueous layer under denaturing
conditions. The release of attached dansylfurfurylamine 3c from
nondenatured BSA at 37 °C was then established by removal of
aliquots and extraction with n-butanol, followed by fluorescence
spectroscopy of the aqueous layer.
The resulting release profiles, likewise found to be curved
(Figure 3), were assigned to two first-order processes as
expected from the adduct half-lives summarized in Table 4.
Thiol adduct half-lives were calculated from the first-order rate
constants determined from the slope of the steeper linear
portion of these graphs, corrected for the rates of amine adduct
release that contribute slightly to the observed data. Amine
adduct half-lives were independently measured from corre-
sponding experiments using unreduced BSA (Figure S6).
Although we have not measured the rate of degradation of
amine adducts as a function of pH, the release is expected to be
pH-dependent. The pKa of adducts like 8 can be quite low due
to pendant ester groups,25 suggesting that both the fast-
observations made above for BME adducts.
We also examined the stability of a representative BSA-5b
conjugate at 23, 30, and 37 °C, showing the thiol adduct to be
4.5 times more stable and the amine adduct(s) to be 11.6 times
more stable, at room temperature vs physiological temperature
(Table S5). Arrhenius activation energies of 20 1 and 32
3
kcal/mol were estimated for thiol- and amine-adduct cyclo-
reversion, respectively. For practical purposes, the more stable
of the BSA−OND adducts may be stored in solution at reduced
temperature for extended periods of time without significant
fragmentation.
OND reagents can also be used to selectively label serum
albumins in complex mixtures. Reagent 1t (75 μM) was
incubated with newborn calf serum (25
Ellman−Wilson assay) at 37 °C. Electrophoretic analysis after
2−30 min of labeling revealed a major fluorescent band near 60
kDa corresponding to OND-labeled BSA (Figure 4, top).
Heating the sample under conditions known to effect the rDA
cleavage of OND−thiol adducts (80 °C, 5 min) resulted in only
partial loss of fluorescence, showing that amine labeling
occurred to a significant extent (as observed for pure BSA)
2 μM in thiol by
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Several popular protein tagging techniques use strong
noncovalent (biotin-streptavidin27) or metal−ligand (oligohis-
tidine−metal28) interactions. In the latter case, the metal ion is
essentially a reversible electrophilic label, cleavable with
imidazole. Both tagging methods have been modified to
incorporate optical signals.29 Additional manipulations are
required to dispose of such labels, such as reductive cleavage
of an electrophilic biotin−diazonium tag,30 or proteolytic
removal of a His tag cleavage with a tag-specific peptidase.31
Fluorogenic OND labels conveniently combine the electro-
phile, cleavage, and detection functions. It has been shown that
thiomaleate 4 and related thiomaleimide8 moieties that remain
on the protein thiols after rDA cleavage of OND-modified
substrates can be cleaved under reducing conditions,32 thereby
rendering the OND modification of protein thiols fully
reversible as well.
The OND family of reagents described here allows for the
convenient tuning of reaction rate, labeling site, and release rate
for the marriage of cargo molecules with serum albumin carrier
proteins.33 By extension, we expect a wide variety of other
scaffolds to be similarly addressable. The ability to control the
rate of fragmentation by altering the substituents on the OND
core and the nucleophile involved in carrier−OND adduct
formation provides flexibility in the design of diverse
degradable biomaterials.
Figure 4. (Top) Gel electrophoresis (1 mM BME, 1% SDS) of
newborn calf serum labeled with OND 1t. Lane 1−4 = labeling with 1t
for 0 (no OND), 2, 6, and 30 min, quenched with BME; lanes 5−8:
samples from lanes 1−4, heated for 5 min at 80 °C. (Bottom) Gel
electrophoresis (1 mM BME, 1% SDS) of human plasma labeled with
OND 5b. Lane 1 = prelabeled BSA; lanes 2−5: human plasma labeled
with 5b for 0 (no OND), 2, 6, and 30 min; lanes 6−9: human plasma
labeled for 30 min, quenched with BME, incubated for 1, 3, 6, and 24 h
at 37 °C, and stored at 4 °C prior to analysis. F = fluorescence
imaging, S = stained (Coomassie), L = molecular weight ladder. The
fluorescence on the bottom of the gels is from small molecule
fluorophores.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental procedures; synthesis of complex OND
esters (Scheme S1), reaction of ONDs with aromatic thiols and
phosphines (Scheme S2), amines (Table S1), UV monitoring
the formation of 4 (Figure S1), dependence of stability of 2 on
alcohol pKa (Table S2), data for release rates vs benzamide
substituents (Figure S2), calculated structure of hydrogen-
bonded thiol adduct (Figure S3), influence of pH on BSA
labeling rates (Figures S4, S5, Table S3), relative reactivity of
nonthiol residues (Table S4), kinetic traces of rDA
and that release from the amine sites was significantly slower. A
similar reaction of OND 5b (75 μM) with human plasma gave
much cleaner labeling of serum albumin (Figure 4, bottom),
consistent with its much greater thiol content (345 20 μM in
thiol). Rapid release of dansyl-furan 7b was observed upon
incubation of the resulting conjugate at 37 °C, consistent with
the expected lability of the thiol adduct.
1
fragmentation of labeled BSA (Figures S6 and S7), and H
and 13C NMR spectra of all new compounds. The material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
mgfinn@scripps.edu
DISCUSSION
■
Notes
We chart here the various modes of reactivity of OND reagents
with proteins under simulated physiological conditions,
focusing on the example of serum albumin attachment and
release. Although the kinetic selectivity for thiols is much
greater than for amines, we find that amine reactivity can be
significant. As demonstrated by successful labeling of thiol-
depleted BSA, temporary and reversible blockage of thiols
makes it possible to direct the OND labeling to nitrogen
nucleophiles.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Science Foundation
(1011796), the NIH (GM083658), and the Skaggs Institute for
Chemical Biology. A.K. is supported by an Eli Lilly Fellowship.
We thank Mr. Jonathan P. Litz for assistance in the early stages
of this project and Mr. Anthony Kislukhin for helpful
discussions.
ONDs are unique among cleavable linkers because the
release function is masked within a potent electrophile, and the
cleavage conditions are exceptionally mild. Reagents that form
mixed disulfides, such as Ellman’s reagent and S-thiosulfo-
nates,26 also combine labeling and release functions, but the
resulting disulfides fail to cleave under the nonreducing
conditions of human plasma. To the best of our knowledge,
no other reaction that forms a covalent bond has been reported
in the context of reversible labeling of plasma components.
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