Glycogen Synthase Kinase 3 (GSK3) is a highly conserved serine/threonine kinase with two paralogs, GSK3α and GSK3β, sharing 85% sequence homology and 98% similarity within the kinase domain . Both isoforms have a large number of substrates 13 and regulate several key signalling pathways, including Wnt/β-catenin, insulin, and mTOR, which are implicated in cellular functions such as proliferation, differentiation, and apoptosis 14 . In the CNS, GSK3β has been linked to neurodegenerative diseases, contributing to Alzheimer's disease (AD) through tau hyperphosphorylation and amyloid-β production, and to Parkinson's disease (PD) by modulating αsynuclein aggregation and dopaminergic neuron survival . GSK3α, while less studied, has been implicated in AD pathology through the promotion of amyloid precursor protein processing and amyloid-β deposition . Given the central role of GSK3 in these diseases, small-molecule inhibitors have been developed with some advancing into clinical trials . However, these efforts have largely failed, it is believed due to either on-target based dose limiting toxicity of complete dual inhibition of GSK3α and GSK3β and/or off-target effects . Further, genetic deletion of GSK3 paralogs has been shown to produce different phenotypes in some models when compared with inhibition . Despite efforts from multiple research groups , to date a rapid, potent and proteome-wide specific GSK3 degrader of chemical probe quality has not been qualified. Such a tool would enable understanding of the potential for kinetically controlled removal of one or both isoforms on the activation status of different GSK3 substrates and the potential to leverage pharmacokinetic/pharmacodynamic disconnects characteristic of degraders , which may differ in effect from occupancy-based inhibition in vivo. Here, we demonstrate a new and potentially generalisable bifunctional molecule synthesis and screening platform, using a concept of 'orthogonally reactive linkers', to identify a chemical probe quality and CNS in vivo active GSK3 degrader directly from a single screen. Developing an orthogonally reactive linker synthesis and screening platform PROTAC potency, kinetics and absorption, distribution, metabolism and excretion (ADME) profiles are all influenced by the choice of ligands, linkers and conjugation chemistry used. We therefore hypothesised that a method whereby all these components could be varied simultaneously would be advantageous to hit finding. Furthermore, with new ligase ligands being disclosed on a regular basis and a desire to explore bifunctional molecules with mechanisms beyond degradation, we reasoned that an approach that did not require pre-conjugation of E3 ligase-linker intermediates would be of benefit. To address this, we proposed a concept of 'orthogonally reactive linkers', molecules that contain two distinct reactive groups, each specifically engineered to selectively react with a unique counterpart in a stepwise fashion (Fig ). We envisioned a streamlined 1-well, 2-step synthesis format such that one end of the linker reacts exclusively with binder A, while the other end selectively reacts with binder B, enabling the sequential synthesis of bifunctional molecules. We aimed to achieve this with methods that could be performed in a plate-based setting, with no protecting groups required and that would result in compounds of sufficient purity that they could be tested directly in cellular screens without the need for purification via chromatography. I.e. amenable to direct-to-biology approaches. Figure . An Orthogonally Reactive Linker synthesis and screening platform concept. A) Proposed approach for simultaneous variation of all components of a bifunctional molecule in a library setting with direct-to-biology cellular screening. B) Selected conjugation chemistries and with strong cation exchange (SCX) semi-purification step. We were interested in focussing our library design and conjugation chemistries such that we could enable best chances of obtaining CNS active GSK3 degraders, for example by limiting hydrogen bond donors, amides and large increases in LogP (Fig ). We also designed linkers with basic centres but avoided strongly basic centres in the ligands themselves. This would allow use of strong-cation exchange (SCX) based catch and release resins to remove starting materials and by-products that may compete with PROTAC mechanism of action in cells 6 . Based on these criteria, we identified SN2 and CuAAC "click" chemistries as a first model system (Fig ). Optimised conditions for this protocol included using DMF as the solvent with Et3N as the base in the absence of additives such as potassium iodide (Fig ). The conditions for the sequential CuAAC step included use of THPTA, which we found was important for basic substrates. To arrive at these conditions, we had performed a proof-of-concept screening to assess the efficiency of the SN2 step under various solvent, base, and additive conditions (Fig ). We designed a linker set encompassing a diverse range of properties, all featuring a secondary amine as the nucleophilic component and avoiding excessive linker length and polarity (Fig ). To maximize the potential for achieving CNS activity in an uncharted property space, we primarily incorporated CRBN-recruiting ligands characterised by low molecular weight, high hydrolytic stability and/or high CRBN affinity . However, given the precedence of VHL penetrant PROTACs for LRRK2 , we also included one VHL-recruiting binder 14 (Fig ). For GSK3 targeting ligands, we designed azide containing building blocks based on PF-367 , 19, and CMP-47, 20 (Fig ). Structural comparisons showed that the triazole in PF-367 engages in a cation-π interaction with R141 and that introducing a triazole at the para position of CMP-47 could enable a similar interaction or, at the very least, avoid disrupting binding affinity. With all library components synthesised (see Supplementary Note for chemistry experimental detail), we generated libraries using a liquid handling system in a 96-well plate format, with identical linker sets and conditions systematically arranged according to GSK3 binder (by plate), linker (by column), and E3-binder (by row). The synthesis began with the SN2 step followed by CuAAC (Fig 2A ) and purification using a 96-well SCX-plate to separate unreacted species from full PROTACs. The eluted products were collected, dried, resuspended in DMSO, and analysed by LCMS for UV purity (Fig ). Our analysis showed that a total of 147 compounds were detected by mass spectrometry, with 103 exhibiting >50% UV purity across the two plates. In addition, the suggests that non-benzyl bromides are potentially less effective than benzyl bromides in the SN2 reaction, and that spirocyclic amine-based linkers displayed poor success rate, likely due to ring opening during SCX. This data, representing the simultaneous variation of multiple GSK3 ligand, E3 ligase ligand and linker types evidence the effectiveness and utility of the proposed orthogonally reactive linker concept for bifunctional molecule library construction.