Intramolecular thiomaleimide [2 + 2] photocycloadditions: stereoselective control for disulfide stapling and observation of excited state intermediates by transient absorption spectroscopy

Thiomaleimides undergo efficient intermolecular [2 + 2] photocycloaddition reactions and offer applications from photochemical peptide stapling to polymer crosslinking; however, the reactions are limited to the formation of the exo head-to-head isomers. Herein, we present an intramolecular variation which completely reverses the stereochemical outcome of this photoreaction, quantitatively generating endo adducts which minimise the structural disturbance of the disulfide staple and afford a 10-fold increase in quantum yield. We demonstrate the application of this reaction on a protein scaffold, using light to confer thiol stability to an antibody fragment conjugate. To understand more about this intriguing class of [2 + 2] photocycloadditions, we have used transient absorption spectroscopy (electronic and vibrational) to study the excited states involved. The initially formed S2 (π1π*) excited state is observed to decay to the S1 (n1π*) state before intersystem crossing to a triplet state. An accelerated intramolecular C–C bond formation provides evidence to explain the increased efficiency of the reaction, and the impact of the various excited states on the carbonyl vibrational modes is discussed.


. General remarks
All chemical reagents and solvents were purchased from chemical suppliers and used as received, without any further purification. Reactions were monitored by thin layer chromatography (TLC), using TLC plates pre-coated with silica gel 60 F254 on aluminium (Merck KGaA), and detected with UV light (254 nm and 365 nm) or KMnO4 chemical stain.
Column chromatography was carried out using a Biotage Isolera using GraceResolv or FlashPure silica flash cartridges. 1 H and 13 C NMR spectra were recorded in the stated solvent on a Bruker Avance Neo 700 instrument equipped with a 5mm helium-cooled broadband cryoprobe operating at 700 MHz for 1 H and 176 MHz for 13 C; or a Bruker Avance III 600 instrument equipped with a 5mm helium-cooled 1 H/ 13 C cryoprobe, operating at 600 MHz for 1 H and at 151 MHz for 13 C. Chemical shifts (δ) are recorded in parts per million (ppm) and coupling constants (J) in Hertz (Hz). The multiplicity of each signal is designated as follows: s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), h (hextet), m (multiplet), or a mixture of these. DEPT, COSY, HSQC, HMBC and NOESY were used to aid the assignment. Infrared spectra were recorded on a Bruker ALPHA FTIR spectrometer with frequencies recorded in reciprocal centimetres (cm -1 ) and absorptions were characterised as follows: s (sharp), br (broad), m (medium), w (weak). Melting points were taken on an Electrothermal IA9000 series apparatus or a Gallenkamp 5A 6797 apparatus and are uncorrected. All mass spectra were obtained at either the Department of Chemistry, University College London on the one of the following instruments: Agilent 6510 QTOF LC-MS system, Finnigan MAT 900 XP double focusing hybrid (EBqQ) mass spectrometer, Thermo Scientific Orbitrap Q Exactive Plus mass spectrometer, Thermo Scientific TRACE 1310 GC-MS connected to Thermo Scientific ISQ single quadrupole mass spectrometer, Waters LCT Premier XE QTOF mass spectrometer; or obtained at the EPSRC UK National Mass Spectrometry Facility (NMSF), Swansea on one of the following instruments: Thermo Scientific LTQ Orbitrap XL linear ion trap mass spectrometer, Waters Xevo G2-S QTOF mass spectrometer. UV-VIS absorption spectra were recorded on a Shimadzu UV-2600 UV-VIS spectrophotometer using a quartz cuvette with a path length of 1 cm and extinction coefficients are reported to 2 s.f. UV irradiation was performed with a medium pressure 125 W mercury lamp (240-600 nm), purchased from Photochemical Reactors Ltd., with the use of a pyrex immersion well. If the lamp was taken out of use, and then reinstalled, the [2+2] photocycloaddition of maleimide to give tetrahydrocyclobuta [1,2-c:3,4-c']dipyrrole-1,3,4,6(2H,5H)-tetraone was performed as a control reaction, 1 prior to irradiation of any unknown compounds.

3-Bromo-1-methyl-1H-pyrrole-2,5-dione, 12 2
To a solution of N-methyl maleimide (556 mg, 5.00 mmol) in MeOH (20 mL), Br2 (0.564 mL, 11.0 mmol) was added dropwise over 10 min. The reaction mixture was stirred for 8 h at room temperature. The solvent was removed in vacuo to give a dark brown oil of the dibromo-Nmethyl succinimide. The oil was redissolved in THF (20 mL) and triethylamine (0.906 mL, 6.50 mmol) was added. The reaction mixture was further stirred for 18 h at room temperature and the solvent removed in vacuo. The crude product was purified by silica flash chromatography

General remarks
All chemical reagents and solvents were purchased from chemical suppliers and used as received, without any further purification. Trastuzumab (Herceptin) was purchased from UCLH Pharmacy in its clinical formulation (lyophilised). This was solubilised in LCMS grade H2O to a concentration of 68.9 μM (10.0 mg/mL). Aliquots of 0.50 mL trastuzumab were stored at -20 °C for up to 6 months. Stock solutions of reagents were made in double-deionised H2O (dH2O), MeCN or DMF as specified, unless otherwise stated. Conjugation experiments were conducted in 1.5 mL Eppendorf tubes at atmospheric pressure at the stated temperature.
Eppendorf 5415 R and VWR Galaxy 14D microcentrifuges were utilised for centrifugation and temperature was controlled by carrying out the reactions using an Eppendorf thermomixer comfort heating block. For the irradiation of Fab conjugates, the samples were placed in a plastic micro UV-cuvette. A 365 nm UVG2 Labino LED torch held at a distance of 14 cm from the sample was used for the irradiation. Ultrafiltration was carried out with Sartorius Vivaspin 500 centrifugal concentrators with a molecular weight cut-off (MWCO) of 10 kDa. UV/Vis absorbance was measured using a ThermoFisher NanoDrop One/One Microvolume UV-Vis Spectrophotometer. These readings were used to determine the protein concentration using the molecular extinction coefficients of ε280 = 215,380 cm -1 M -1 for full antibody and ε280 = 68,590 cm -1 M -1 for Fab. 6 The following buffers were made using dH2O; the pH adjusted using

Irradiation of Fab conjugate 6
Fab conjugate 6 was adjusted to 40.0 μM, 70.0 μL in PBS pH 6.0. This was then irradiated for 2 min to give hydrolysed conjugate 7, observed by LCMS.

Ab initio calculations
The model thiomaleimide shown in Figure 10 was chosen for ab initio calculations to reduce computational cost while still capturing the main features of the molecule. The structure of the ground state molecule was optimised using DFT with the CAM-B3LYP functional and the 6-311++G basis set in Gaussian 09. 8 Frequency calculations were performed to confirm that a minimum had been achieved. The optimised atomic positions are given in Table 3. This optimised structure was then used for calculations of the excited states with the EOM-CCSD method and the 6-311++G** basis set in Q-Chem 5.2. 9 CAS-PT2 calculations were also performed on the model chromophore with the 6-311++G** basis set and an active space with 12 electrons and 8 orbitals in MolPro. 10,11 The active space included six π-orbitals and two n-orbitals with O lone pair character and these MOs are shown in Figure   11. Vertical singlet and triplet excitation energies E and oscillator strengths f for the model thiomaleimide in Cs symmetry calculated using both the EOM-CCSD and CAS-PT2 methods are listed in Table 4.  Table 4: Calculated energies E, oscillator strengths f in parentheses and corresponding wavelengths for electronic transitions from S0 for the model chromophore in Cs symmetry using (a) the EOM-CCSD method with a 6-311++G** basis set and (b) the CAS-PT2 method with a 6-311++G** basis set and an active space with 12 electrons and 8 orbitals. Note that the oscillator strengths reported alongside the CAS-PT2 energies are from the CAS-SCF calculation. Figure 11: Molecular orbitals (MOs) included in the active space for the model chromophore. These are generated from ab initio calculations using the CAS-PT2 method, performed using MolPro 10,11 with the 6-311++G** basis set and an active space with 12 electrons and 8 orbitals. Orbitals n1, π1 and π1* are involved in the formation of the S1 (n1π1*) and S2 (π1π1*) excited states.

Quantum yields
For quantum yield measurements, a fluorescence quartz cuvette placed in a sample holder on a magnetic stirrer was used to provide homogeneous irradiation of the whole sample. The sample was irradiated using a 365 nm UVG2 Labino LED torch held at a distance of 38 cm from the cuvette. The UV absorption was measured simultaneously using an Ocean Optics HR 4000 spectrometer. The spectrometer beam was at a perpendicular pathway to the LED torch beam. The background was measured using a blank sample of MeCN or DCM, as required. The power and photon flux of the LED reaching the cuvette was calculated using onitrobenzaldehyde as a chemical actinometer, with a known quantum yield Φ of 0.43 ± 0.02. 12 The following equation was used to calculate the LED torch's photon flux, I0 (M s −1 ): kfit is the decay constant (s −1 ) calculated by fitting a mono-exponential growth function to the absorbance vs. time graph at of the reaction of o-nitrobenzaldehyde at 283 nm (example in Figure 12); c0 is the initial concentration used (M); Φ = 0.43 ± 0.02 is the quantum yield; and Ai is the initial absorbance at the 365 nm irradiation wavelength.
The power of the LED torch, P was calculated from the following equation: h is Planck's constant ( (3) r is the initial rate of change in concentration (M s −1 ) measured by the gradient of the concentration vs. time graph up to ~10% conversion (to keep the absorbance at the irradiation wavelength constant) (example in Figure 13); 13 and Iabs (M s −1 ) is the amount of photons absorbed by the sample. This is calculated from the equation: The value of Φcycloaddition = Φreactant for the intramolecular reactions, but Φcycloaddition =     were recorded using a spectrometer (Avantes, AvaSpec-ULS1650F) for TEAS and using a Horiba iHR320 imaging spectrometer with a mercury cadmium telluride detector array for TVAS. The delay between the pump and probe pulses was controlled using a 500 mm delay stage, giving a maximum delay of 2.5 ns. The TEAS data was chirp-corrected using the KOALA package. 14 Regions of the TEAS and TVAS spectra where residual pump or ground state bleach (GSB) obscured the data were removed prior to analysis and these false colour plots are presented in the main text. The original false colour plots showing signal masking are shown in Figure 14. were not included in the global analysis due to interference from the GSB and residual pump in this region. For the TVAS experiments, the global analysis was only conducted between 1560 and 1630 cm −1 as this region contained the key bands. It was not feasible to include the band at 1665 cm −1 due to very few data points at longer timescales. The IRF values are summarised in Table 5 and the Decay Associated Spectra (DAS) are shown in Figure 15.

S50
The spectral profile at different timepoints for the TEAS data is shown in Figure 16. Bisthiomaleimide 4a shows longer time delay features for the bands at 585 and 385 nm, which is not observed for thiomaleimide 1.