Frequenters of the Open Flask may
have noticed the increased occurrence of peptide substrates in some of our
recent methods reports. Nearly two years ago, we were joined by an
exceptionally talented post-doctoral researcher, Dr. Lara Malins; her expertise
in the field of peptide synthesis has proven a boon for the group, and her
contributions can be found scattered throughout many of our recent papers. “Peptide Macrocyclization Inspired by
Non-Ribosomal Imine Natural Products” (J.Am. Chem. Soc. 2017, ASAP) however, represents the Baran Lab’s
first true foray into the field of peptide chemistry.
While the project has been
rewarding, it was not without its practical challenges. As an historically
total synthesis-based group, we are not equipped with the standard supplies and
instrumentation found in even the smallest of peptide synthesis laboratories;
instead we have relied on both the generosity of other TSRI groups (shout out
to the Ghadiri lab!) and our own creativity to accomplish this study. Reactions
that required heating—specifically, the loading of amino aldehydes onto our
manually prepared Rink TG resin—posed a unique problem, as our orbital shakers
are limited to room temperature agitation. As such, we were forced to use rubber bands
and rotavaps, sans vacuum, to simultaneously stir and heat the reaction
vessels. While this was the best method available, it wasn’t great… We fished soaking
syringes out of the water bath on more occasions than we care to remember. We
were also on the receiving end of judgement from labmates about (a) what on earth we thought
we were doing and (b) whether this was really the best use of a rotavap…
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“I
think your peptide is dry, Justine…” – fellow labmate after rotovap
resin-loading for over 5 h…
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It is also worth mentioning that the majority of
substrates were made without the aid of an automated peptide synthesizer,
relying instead on our own two (four) hands. Scale-up for initial reaction
screening meant running multiple 300–400 μmol
scale reactions in parallel; tennis elbow has nothing on peptide elbow!
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When
Phil asks for a gram-scale reaction, you find a way…
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After all of the work (and
swollen elbows), we were thrilled to submit a JACS communication in the fall of 2016, wherein we described a
novel approach to thermodynamic peptide macrocyclization. This unconventional
method allows for late-stage diversification of high-value substrates by virtue
of an inherently reactive imine intermediate, a process inspired by a
non-ribosomal peptide synthetase/reductive release mechanism. Incorporation of
isotope labels and bioorthogonal functional handles proved facile, as did
further manipulation of the installed moieties. Additionally, four distinct
natural products and associated analogues were synthesized, each in a time
frame of about 2 days (start to finish). As per Baran Lab dogma, the reactions require no protecting groups and are tolerant of all proteinogenic
functionality.
While much of the initial
feedback was positive, we received a few valid critiques to address prior to
resubmission. The first point raised concerns over whether conformational
predisposition of the chosen amino acid sequences contributed to the robust
protocol. Given that many of our sequences were inspired by natural products,
we held similar concerns; in the full paper (out today), we opted to use an
online random sequence generator to minimize the possibility of practitioner
bias in sequence selection. We were pleased to discover no notable difference in reaction rate or yield between our original
substrates and those picked at random. Our observations were further
corroborated by variable temperature NMR studies done by our friends at
Bristol-Myers Squibb (thanks, Kevin and Paul!). Unsurprisingly, Kevin was able
to identify hydrogen-bonding networks in the four natural products synthesized,
which likely contribute to the facility of cyclization in these cases. In
contrast, the other substrates investigated showed few, if any, of these interactions.
While we cannot claim that conformational bias plays no role, we have
determined that it is not a strict prerequisite.
A second, but related, matter
raised by the referees was the origin of selectivity in sequences that possess
an internal lysine residue (i.e., cyclization at the terminal amine versus the lysine ε-NH2). In our
original report, we had included only one example with an internal lysine,
which—as pointed out by one of the reviewers—was an consideration that should be further
explored. We agreed, and have now expanded our study to include six examples,
one of which was generated at random, and are confident that reaction conditions, regardless of protocol,
can be successfully tuned to favor terminal amine cyclization. Once again,
VT-NMR studies suggest no internal bonding networks in the lysine-containing
peptides analyzed, indicating that conformational bias is not the sole
contributing factor.
We are immensely grateful for the
insightful criticism received from our first submission—the suggestions
provided helped guide our revision process, and served as a prime example of
peer-review at its finest. We now present a thorough, and much expanded, study
on our approach to thermodynamic peptide macrocyclization. Enjoy!
Justine
