Ψ

August marks the start of the academic year here at TSRI, and with every new semester I become a bit retrospective about my own first year of graduate school. Before I joined the Baran Lab, I was certain that I had a handle on the types of chemistry the group pursued: small-molecule secondary metabolites, usually with a twist. I arrived determined to work on a Baran standard. Instead, Phil pointed me toward a perspective written by Professor Albert Eschenmoser in which he laments the fact that, though a decidedly natural products problem, the synthesis of DNA had gone virtually ignored by natural products chemists (Tetrahedron 2007, 63, 12821). Arguably the most important molecule in natural history and we, as a community, missed it. 

The DNA structure problem, which had been attacked and solved by non-chemists, and … revealed itself as …the major problem that nature had in store for natural product chemistry. – A. Eschenmoser (2007)

This quote became a quiet mantra of sorts, driving much of my interest in the synthesis of biomolecules (see: macrocyclization,CITU,peptide functionalization, and more musing on peptide functionalization). Then, about two years ago, a variation on the problem was presented to us by collaborators at Bristol-Myers Squibb: Can we improve the synthesis of medicinally-relevant oligonucleotides? 

Despite our collective lab ignorance around oligonucleotide synthesis, a remarkable graduate student, Kyle Knouse, was brave enough accept the challenge. It’s important to emphasize here just how courageous this choice was: The lab had no experience and no equipment with which to rewrite the rules of oligonucleotide synthesis. 

Given my stated interest, I was asked to join the project and—together with Drs. Martin Eastgate, Mike Schmidt, and the BMS team—we began to survey the existing literature.

[left](L to R): Dr. Chao Hang, Dr. Antonio Ramirez, Dr. Ye Zhu, Dr. Mike Schmidt, Dr. Bin Zheng, Dr. Jason Zhu, Dr. Matt Winston, Dr. Changxia Yuan. Not pictured: Dr. Ivar Mcdonald, Dr. Rick Olson, Dr. Stephen Mercer, Dr. Qinggang Wang, and Dr. Peter Park. [right] Dr. Martin Eastgate. 

We discovered that while the earliest reports of nucleotide synthesis relied on the natural phosphorous (V) oxidation state, the late 1970s saw the lethargic P(V) largely supplanted by P(III) in the form of phosphoramidites and H-phosphonates. Perhaps most striking was the apparent reliance on incremental modifications to advance the field; the P(III)-based phosphoramidite approach remains the standard mode of construction to this day.  

Had we any expertise, we almost certainly would have followed suit. For most, P(III) represented the most practical way to assemble oligonucleotides; to us, it was an unnecessary shackle. Liberated by our naivety, we chose instead to approach the problem as we would any other natural product: identify the most logical disconnections, limit concession steps, and aim for ideality. 

Adding yet another layer of complexity, both teams were specifically interested in P-chiral phosphorothioate oligonucleotides. We set forth with an ever-expanding list of requirements: the platform need be inexpensive, bench-stable, modular, and simple to purify; it must operate under complete stereocontrol, with near-equal access to both enantiomers of the chiral backbone; and, finally, it should feature a generality able to tame even the most discriminating of deoxyribonucleoside monomers. 

Building on chemistry developed in Stec’s lab, the team synthesized a P(V)-reagent in one step from limonene oxide, which served as the chiral scaffold. The original approach, while creative, fell short in several important ways; Stec himself cited the tedious reaction protocols, difficult diastereomer separations, and operational complexity as barriers to widespread use. 

The Stec Precedent

Interest from BMS continued to grow as the project progressed, and we were soon approached with the possibility of yet another application—Could the nascent BMS-Scripps reagent platform be designed with cyclic dinucleotides in mind? While there is much to be said about the medicinal potential of CDNs, I am not the person to do it; instead, I’ll direct readers to a fantastic C&EN article published earlier this year. From an academic perspective, however, we saw an opportunity: this alluring scaffold promised a challenging and underexplored class of natural products. Indeed, most CDN syntheses are buried deep within the patent literature and published without the rigor expected of a proper total synthesis campaign. As with many complex natural products, current approaches resemble a navigational land war of protecting groups and redox manipulations, not to mention the unassailable phosphorous stereocenters. Until now, synthesis of a single, pure CDN analogue took weeks. 

With another goal now in mind, both teams (the Scripps side now expanded by two spectacular post-docs, Julien and Cian) worked tirelessly and collaboratively to construct what would eventually become the phosphorous–sulfur incorporation (PSI or Ψ) reagent platform, the full details of which can be found in the accompanying manuscript. We hope you’ll agree that our mission was accomplished: PSI chemistry is practical, simple, and offers complete, predictable control over the phosphorous stereochemistry. Complex CDNs can now be synthesized in two days’ time as a single diastereomer, a point previously unimaginable. Access to diastereomerically-pure oligonucleotides has never been easier; the machine confines required of sensitive P(III) chemistry are a thing of the past.   

We leave you with a question posed by one of the giants of phosphorous chemistry, Frank Westheimer (Science 1987, 235, 1173): Why did Nature choose phosphates? He notes in the cited exposition that phosphates are found everywhere and can do almost everything—“genetic tape” is the colloquialism he seems partial to. By contrast, chemists have been reluctant to employ this all-important functional group with similar abandon. We respectfully suggest that the interesting question as it pertains to practicing chemists is not, “Why did Nature select phosphates?”, but rather, “Why didn’t we?” 

—  Justine and Team Oligo

p.s. Ψ reagents will soon be commercially available from Sigma-Aldrich