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One example involves the preparation of bispecific antibodies by
generating Fab fragments with a p-acetylphenylalanine UAA, cap-
able of undergoing oxime ligations forming therapeutically useful
bioconjugates.39,41
Due to the well-documented utility of both bioconjugates and
photocaging, we sought to combine the two techniques into a sin-
gle unnatural amino acid. The gap in the current bioorganic tech-
nology prompted our interest in developing such a bifunctional
UAA. Ideally, this UAA would harbor a handle for bioconjugation
and also facilitate a degree of conjugate flexibility, allowing it to
disproportionate upon light irradiation.42 These properties may
be especially useful in the delivery of cytotoxic molecules via con-
jugation to antibody targeting agents that may alter the efficacy of
the molecule due to the relative size of the antibody.41,43 Upon
delivery and endocytosis to the cell, the small molecule could be
photocleaved to remove the potentially inactivating steric bulk of
the antibody allowing it to achieve its full activity (Fig. 1). A purely
photocleaving UAA without the capacity for bioconjugation has
previously been developed and utilized to cleave the protein back-
bone upon light exposure.44 Our envisioned UAA expands upon
this feature to introduce a handle for a bioorthogonal conjugation
reaction.
Toward this goal a novel UAA was developed containing both a
dimethoxy-ortho-nitrobenzyl functionality for photoreactivity,
and an alkynyl moiety for conjugation (Fig. 1). UAA 1 could be syn-
thetically accessed beginning from commercially available vanillin
(2) in a concise six-step sequence (Scheme 1). Vanillin was first
propargylated to form 3 in order to provide the alkyne functional-
ity for future bioconjugation. The nitro group was then installed
using nitric acid to afford 4 in moderate yield and high regioselec-
tivity. The aldehyde 4 was then reduced with sodium borohydride
in 1 M NaOH to afford the alcohol 5 in 71% yield. Interestingly,
reactions in ethanol, a more conventional solvent for this type of
reduction, resulted in substantially lower yields even when used
in large excess, necessitating the reduction in aqueous sodium
hydroxide. The alcohol 5 was then brominated with PBr3 to provide
6, which was then alkylated with diethyl acetamidomalonate to
yield the protected UAA 7. Several conditions were examined for
the conversion of 6 to 7; however, the most efficacious reaction
progressed under microwave irradiation.45 Thus, 7 was depro-
tected in 6 N HCl under microwave irradiation to yield the desired
UAA in an overall yield of 19%. The purity and identity of 1 was
confirmed by both NMR and GC/MS analyses.
Scheme 1.
into a protein. Typically, a double-sieve selection on an aaRS library
is required to identify a functional aaRS; however, recent reports
have found several existing aaRSs that displayed promiscuity
toward the incorporation of additional UAAs.46,47 We hoped to
exploit this promiscuity to obviate the necessity of an aaRS selec-
tion, and thus initiated a screen with several existing synthetases
for the incorporation of 1 using a GFP reporter (sfGFP; super-fold-
ing green fluorescent protein). The ability to incorporate the UAA
would result in a fluorescent signal due to the production of func-
tional GFP, whereas a failure to incorporate results in a non-fluo-
rescent truncated protein due to the inability to suppress the
TAG stop codon. Thus, increased fluorescence could be correlated
to aaRS promiscuity toward 1. A range of aaRSs were selected
including Bipy, pCNF, ONBY, NapA due to either their previously
reported promiscuity, their large UAA binding pocket, or their abil-
ity to incorporate structurally similar UAAs. Gratifyingly, upon
screening the pCNF aaRS, a significant increase in fluorescence in
the presence of 1 relative to the absence was observed (see Sup-
porting Information). Consequently, the pCNF-aaRS was selected
for expression of 1. To confirm incorporation, cells harboring a
plasmid that encodes the aaRS/tRNA pair and a plasmid encoding
GFP with a TAG mutation at surface exposed residue 151 were
induced with IPTG/Arabinose in the presence of 1, and incubated
16 h at 30 °C. The cells were then lysed and GFP was purified using
a commercially available Ni–NTA resin and analyzed via SDS–PAGE
and fluorescence (Fig. 2). Based on both fluorescence data and gel
analysis, 1 is incorporated into GFP at 68% relative to wild type
GFP expression.
With the desired UAA in hand, it was necessary to elucidate an
appropriate aaRS that could recognize and effectively incorporate 1
Following expression and purification of the GFP containing 1, a
proof-of-concept experiment was performed to ascertain the feasi-
bility of both the bioconjugation and the photoreactivity of the
UAA. Our laboratory recently reported a Glaser–Hay bioconjuga-
tion involving the coupling of terminal alkynes.37,48 Consequently,
this novel bioconjugation was employed with the new UAA based
on the alkynyl handle 1. The mutant GFP was reacted with an
alkyne containing Alexafluor-488 molecule in the presence of
Figure 1. Strategy for photoregulation of protein conjugates. Once incorporated
into a protein context the alkynyl handle facilitates either a Glaser–Hay reaction or
a 1,3-dipolar cycloaddition to generate a bioconjugate. Irradiation with UV light
then facilitates protein degradation and delivery of the bioconjugate partner. The
red and blue represent the protein backbone, which upon cleavage is degraded.
Padilla, Marshall S.
