Pentafulvene–Maleimide Cycloaddition for Bioorthogonal Ligation

Abstract

The applications of bioconjugation chemistry are rapidly expanding, and the addition of new strategies to the bioconjugation and ligation toolbox will further advance progress in this field. Herein, we present a detailed study of the Diels–Alder cycloaddition (DAC) reaction between pentafulvenes and maleimides in aqueous solutions and investigate the reaction as an emerging bioconjugation strategy. The DAC reactions were found to proceed efficiently, quantitatively yielding cycloadducts with reaction rates ranging up to ∼0.7 M–1 s–1 for a series of maleimides, including maleimide-derivatized peptides and proteins. The absence of cross-reactivity of the pentafulvene with a large panel of functional (bio)­molecules and biological media further demonstrated the bioorthogonality of this approach. The utility of the DAC reaction for bioorthogonal bioconjugation applications was further demonstrated in the presence of biological media and proteins, as well as through protein derivatization and labeling, which was comparable to the widely employed sulfhydryl–maleimide coupling chemistry.

Injectable Diels–Alder cycloaddition hydrogels with tuneable gelation, stiffness and degradation for the sustained release of T-lymphocytes

“…Determination of the pseudo-first order rate constant for hydrolysis of the maleimide groups ( k obs = 2.89 × 10 −6 s −1 ; t 1/2 = 2.8 d) was consist with previously report values, 62 and confirmed that the timescale of maleimide hydrolysis is insignificant compared to the fulvene–maleimide DAC. 54 In comparison, for PEG 10k -Ful 4 , no deterioration of the fulvene groups was detected over a period of 12 d, indicating that they are stable in PBS, although 9% of ester linkages hydrolysed during this period as evidenced by the appearance of signals resulting from 4-(cyclopenta-2,4-dien-1-ylidene)pentanoic acid (Fig. 1b).…”

“…53 Towards faster gelling DAC systems our group recently investigated the kinetics of the fulvene–maleimide coupling pair in aqueous systems, which displayed an order of magnitude faster kinetics than the furan–maleimide DAC. 54 The application of this DAC chemistry has also been reported by Madl et al , who prepared PEG-based hydrogels for cell encapsulation and culture that effectively prevented cell sedimentation and remained stable for several months under physiological conditions. 55 However, for the delivery and release of ACTs it is desirable to have fast gelation kinetics to minimise cell sedimentation as well as the sustainable release of the cells from the hydrogel matrix over a desirable period of days to weeks.…”

“…4-(2,4-Cyclopentadien-1-ylidene)-pentanoic acid and 3-(2,5-dioxo-2,5-dihydro-1 H -pyrrol-1-yl)propanoic acid were prepared as reported previously. 54 Nylon mesh with 120 μm pore size was purchased from Lincraft (Australia). PolyLite TM and Hytrel® 3D printing filaments were purchased from 3D Printing Solutions (Australia).…”

“…These results are consistent with our previous studies that indicate 6,6-dialkyl pentafulvene derivatives display excellent stability in aqueous solutions (pH 6-9) and various media. 54…”

“…Interestingly, rheological measurements for gelation of the DACgel % -A series in complete X-Vivo medium revealed an increase in the T gel as compared to measurements in PBS for DACgels prepared at 7.5 and 10% w/w, which may be due to the presence of thiolfunctionalised proteins in the medium consuming a fraction of the maleimide end-groups and reducing the DAC reaction rate. 54 To confirm that hydrogel formation occurred through fulvene-maleimide DAC and determine the endo-exo stereospecificity of the reaction, the formation of DACgel 5 -A in deuterated PBS was monitored via NMR spectroscopy at 20 1C (Fig. 2).…”