High Rates of Quinone-Alkyne Cycloaddition Reactions are Dictated by Entropic Factors

: Reaction rates of strained cycloalkynes and cycloalkenes with 1,2-quinones were quantified by stopped flow UV-Vis spectroscopy. Surprisingly, it was determined that 8-membered BCN-OH reacts substantially (16 times) faster than the more strained 7-membered THS. Thermodynamic activation parameters, obtained from the linearized Eyring equation and stopped-flow measurements, revealed that the reaction of both BCN-OH and THS with ortho -quinones is entropy-controlled, ruling out a relevant contribution of secondary orbital interactions (SOIs) as earlier proposed. The strain-promoted oxidation-controlled ortho -quinone (SPOCQ) cycloaddition is an oxidation-inducible [4+2] cycloaddition of a 1,2-quinone with a strained cyclic alkyne or alkene that follows an inverse electron-demand Diels – Alder (IEDDA) reaction mechanism. 1,2 Due to its high rates and efficiency, SPOCQ chemistry has been successfully employed for the chemical modification of surfaces and for the preparation of bioconjugates. 3 – 6 In comparison to the widely applied strain-promoted azide-alkyne cycloaddition

The strain-promoted oxidation-controlled ortho-quinone (SPOCQ) cycloaddition is an oxidation-inducible [4+2] cycloaddition of a 1,2-quinone with a strained cyclic alkyne or alkene that follows an inverse electron-demand Diels-Alder (IEDDA) reaction mechanism. 1,2][9][10][11] Whereas details of the mechanism and rate constants of SPAAC are well-established, 12,13 in-depth investigations into SPOCQ are scarce and mostly limited to computational studies. 14n the current paper, the experimental determination of second-order rate constants and a detailed computational analysis of SPOCQ between a model o-quinone and various strained alkenes and alkynes is described. We also quantified the effect of derivatization of two of the most commonly employed strained unsaturated systems, BCN (endo-bicyclo[6.1.0]non-4-yne)and TCO (trans-cyclooctene), on the associated second-order rate constants to translate our findings to derivatives used in conjugation chemistry.
][19][20][21] The model SPOCQ reaction we investigated is depicted in Scheme 1a.Specifically, we employed the relatively stable 4-tert-butyl-ortho-quinone 1, which has a distinct optical absorption maximum at 395 nm that allows an easy spectroscopic analysis of the reaction rates.Our matrix of unsaturated carbon-carbon bond-containing compounds 2-7 covers a range of ring sizes, associated strain and relevant derivatization (Scheme 1b).Scheme 1.(a) Generalized scheme for the SPOCQ reaction.(b) Matrix of strained unsaturated carbon-carbon bond containing reagents that were studied.Firstly, we compared the recently published THS 2 -a highly strained 7-membered alkyne with unprecedented reaction rate constants in SPAAC reactions with azides 22, 23 -with BCN-OH 3, an 8-membered alkyne that is currently one of the benchmark reagents for metal-free click chemistry applications. 248][29] Importantly, as all of these strained unsaturated systems are usually not applied as such, but incorporated into a functionalized construct, we also determined the effect of alcohol carbamoylation on the rate constants of BCN and TCO (with compounds 4 and 6), respectively.
Accurate determination of the reaction rates and second-order rate constants of the SPOCQ reaction was performed by stopped-flow UV-Vis spectroscopic analysis at 25 °C in a methanol-water (1:1) mixture (for set-up see Supporting Information). 30Pseudo-first-order reaction conditions in which 10-100-fold excess of the strained systems 2-7 with respect to o-quinone 1 were used.The concentration-independent second-order rate constant k2 was derived from the slope of the k′ (or kobs) versus [B]0plot (with [B]0 equaling the starting concentration of the excess reagent, Figure 1).Notably, the SPOCQ rate constant for BCN-OH 3 of 1824 (± 16) M -1 s -1 is substantially higher than previously reported values (496-1112 M -1 s - 1 ), 1,7 which may be rationalized by the fact that sub-optimal conditions were employed previously (e.g., close to equimolar ratios, suboptimal mixing, inability to measure conversion within seconds after mixing).Secondly, we surprisingly found that the k2 value for THS 2 of 110.6 (± 2.3) M -1 s -1 is approximately 16 times lower than that of BCN-OH 3, which is in striking contrast with the reported observation that THS 2 reacts at least 5 times faster than BCN-OH 3 in cycloaddition with azide (i.e., SPAAC).
In line with our earlier observations, strained alkenes undergo slower cycloaddition with o-quinone than strained alkynes.Specifically, TCO-OH 5 with a k2 value of 11.56 (± 0.11) M -1 s -1 is 10-fold less reactive than THS 2 and 160-fold compared to BCN-OH 3.With respect to the influence of the chemical derivatization, conversion of the hydroxyl group of the well-established probes BCN and TCO into a carbamate lowered the rate slightly (26-44%), i.e., to 1354 (± 19) M -1 s -1 for BCN-carbamate 4, and to 6.47 (± 0.04) M -1 s -1 for TCO-carbamate 6. Surprisingly, the k2 value of 8.36 (± 0.14) M -1 s -1 for cyclopropene-derived probe 7 is somewhat higher than that of TCO-carbamate 6, suggesting that cyclopropene derivatives are potentially a preferred class of strained alkenes for application in SPOCQ chemistry, specifically when a small ring size is preferred, such as in crowded environments.2][33][34] To this end, the k2 values were determined at 5 °C, 13 °C, 21 °C, 29 °C and 37 °C, respectively (Figure 2).This range was chosen as the lower temperatures represents values used for the preparation of protein conjugates such as ADCs, while the highest temperature is relevant for potential in vivo applications.As a result, the reaction of o-quinone 1 with THS 2 is associated with a ΔH ‡ of 0. Clearly, the enthalpy required to form the TS for the reaction between 1 and either BCN-OH 3 or THS 2 is only minimal.Considering that the kinetic energy kT at room temperature translates to 0.6 kcal/mol, our data does not provide evidence for any significant contributions of secondary orbital interactions -which are by definition enthalpic interactions-, as was recently proposed for the SPOCQ reaction between BCN-OH 3 and o-quinone 1. 15,16 In contrast, the investigated SPOCQ reactions are by and large entropy-controlled reactions.Specifically, the large negative ΔS ‡ values prove that both reactions are associative in nature and require precise positioning of the reagents to form the transition states.In addition, more order needs to be imposed on the reaction of THS 2 than on that of BCN-OH 3, which we tentatively explain by considering that the TS formed during approach of THS 2 to the plane of 1 has to accommodate the steric bulk of the four methyl groups next to the alkyne.This difference in entropy at 25 °C between 2 and 3 (difference in TS ‡ = 3.2 kcal/mol) apparently outcompetes the difference in enthalpic factors (-1.5 kcal/mol) to yield an overall difference in Gibbs energy of activation at 25 °C of 1.7 kcal/mol in the advantage of BCN-OH 3 SPOCQ over THS 2 SPOCQ.
To further support our empirical findings, computational investigations on the TS were performed using the M06-2X functional with the 6-311+G(d,p) basis set and including the implicit solvent model SMD. 35To this end, the free energy barriers for the cycloaddition of TCO, endo-BCN, THS, and CP were calculated for the reaction in water and in MeOH.The transition state structures and the calculated activation free energies (ΔG ‡ ) and reaction free energies (ΔGRXN) are displayed in Figure 3.As observed, the activation free energies for these SPOCQ reactions range from 15 to 20 kcal/mol, in reasonable agreement with experiment, given the non-explicit nature of our solvent model.In all cases, the reaction proceeds via a non-synchronous transition state with C•••C distances ranging from 2.22 Å to 2.30 Å.In agreement with experimental results, the computed rate constants predict that BCN will react appreciably faster with o-quinone 1 than THS and TCO, with a factor 4 and 50 times, respectively.
In conclusion, stopped-flow UV-Vis spectroscopic analysis of the cycloaddition of 4-tert-butyl-ortho-quinone 1 first of all revealed that the novel strained alkyne THS 2 reacts 16 times slower than BCN-OH 3 in SPOCQ.Also, we find that derivatization of the strained warhead reduces this rate slightly.Most importantly, experimentally quantified thermodynamic activation parameters showed that the reaction of THS 2 and BCN-OH 3 with o-quinone 1 is essentially an entropy-controlled reaction, which contradicts the proposed involvement of secondary orbital interactions in SPOCQ chemistry.

ASSOCIATED CONTENT
Supporting Information.Details on the synthesis and characterization of THS 2 and carbamate derivatives 4 and 6, the setup used for the stopped-flow experiments, data on all kinetic measurements, derivatization of mathematical equations and specifics of the computational studies.This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 3 .
Figure 3. Transition state structures, Gibbs activation free energies, and Gibbs free energies of reaction for the SPOCQ of BCN, THS, TCO and CP (top to bottom), with quinone 1.Bond lengths are shown in red in Å and energies are given in kcal/mol.