Degradable Conjugates from Oxanorbornadiene Reagents

Synthesis and Stability of OND–Thiol Adducts

Oxanorbornadienes 1 were readily obtained from inexpensive furans and dial-kyl 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 of furfurylamine, as a free base or protected as various salts (trifluoroacetate, tosylate), invariably gave complex mixtures resulting from conjugate addition to dimethyl acetylenedicarboxylate. 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 obtained 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 bridge-head 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.

Table 2.

Half-lives of 1,4-disubstituted thiol adducts 6.

Adduct	R1	regioisomer ratio	half-lifea
6a	Me	–	<1 h
6b	CH2NHDansyl	2:1	1.9 ± 0.1 h
6c	CH2NHCOC6H4pNO2	1.3:1	3.1 ± 0.2 h

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^a

Measured in CDCl₃ by NMR.

The decomposition of oxanorbornene adducts 2 was folowed by ¹H 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 hours. 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 furfurylamine 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, 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 pK_a 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 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 non-selectively, 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 two hours 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 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 non-tertiary amines with ONDs were studied in 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 equivalents 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.

Substantial differences in stability were observed for these molecules. Anti-8 disappeared within 7 days at room temperature, 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, adduct 9 showing a half-life of approximately 12 hours. 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 (t_1/2 ≈ 280 and 24 days, respectively). Interestingly, protonation (by addition of 2 equivalents of trifluoroacetic acid) of adducts 8 and 11 made them less stable than the parent amines, to a much greater degree for anti-8•ETFA (t_1/2 < 1h) and anti-11•ETFA (t_1/2 ≈ 1.9 h) than for syn-8•ETFA (t_1/2 ≈ 90 d) and syn-11•ETFA (t_1/2 47 d). In contrast, the anti- and syn-isomers of 12•ETFA showed a much smaller decrease in stability relative to the free base (t_1/2 ≈ 1.2 d and 15 d, respectively).

Stability Trends

It is apparent that the stability of OND adducts is governed by several stereoelectronic factors. Four trends can be identified from the above data.

1. Thiol adducts (2, 6) undergo retro-Diels–Alder fragmentation 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 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 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 hyperconjugative n→σ* interaction by increasing C4-C3-X angle. Potential transition state stabilization by the second methyl cannot account for such 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 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 decreasing acidity of parabenzamide 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 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 non-thiol 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 thiolate anion with an estimated second-order rate constant of 8540 M^–1s^–1 (Table 3) vs. a reported value of 14200 M^–1s^–1 for an N-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^–1s^–1, again similar to the reported reaction of N-ethylmaleimide with N-terminal valine under the same conditions (k_obs = 0.006 M^–1s^–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 k_Nu and k_rel(Nu), Table 3), the model lysine amine was 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 attachment 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 brief treatment with dithiothreitol (DTT)²⁴ and purified by size-exclusion filtration. The resulting reduced BSA (rBSA) exhibited 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 2^nd-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 k_amines 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.

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 rates to the calculated protonation state of a single amine nucleophile gave an apparent pK_a of 7.9 for this reaction site, a value well within the possible range of lysine side chain pK_a (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 labeled with a moderate excess of fluorogenic OND reagents under conditions designed to promote complete alkylation of available thiol and a modest level of reaction with protein 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 non-denatured 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 1^st 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 corresponding 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 pK_a of adducts like 8 can be quite low due to pendant ester groups,²⁵ suggesting that both the fast-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 decrease in thiol adduct half-life (1u vs. 1v), and (c) BSA-amine adducts were generally found to be more stable than BSA-thiol adducts. Each of these findings correlated well with 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) 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 cycloreversion, 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 ± 2 μM in thiol by Ellman–Wilson assay) at 37 °C. Electrophoretic analysis after 2–30 minutes 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) 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.