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The trimethylsilyl protecting group on the acetylene group is retained, as 4-ethynylphenol (contrary to its methoxy congener 4-ethynylanisole) is highly unstable. Alkylation of the phenol moiety to an ether group eliminates the instability, thus enabling removal of the TMS group.
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The non-regiopure conjugate was generated analogously to its regioselective γcongener by only using a non-regioselective methotrexate PEG azide, prepared as described in the Supporting Information.
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