This revolution can be traced back to a single paper published in 1981 in an organic-chemistry journal called Tetrahedron Letters. The paper, "Deoxynucleotide phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis," (Tetrahedron Lett., 22[20]:1859-62, 1981) was written by Serge L. Beaucage and Marvin H. Caruthers and has since been cited nearly 1,400 times. It was the breakthrough paper on oligonucleotide synthesis that launched Beaucage on a career that has now included another eight papers cited more than 100 times each.

Beaucage, 48, earned his Ph.D. degree in chemistry under the tutelage of Kelvin Ogilvie at McGill University 1979 and then spent two years as a post-doctoral fellow of the National Research Council of Canada (NRCC) with Caruthers at the University of Colorado. In 1988, after stints with Smith-Kline Beckman and Stanley N. Cohen at Stanford University School of Medicine, he joined the Food and Drug Administration. He is now a research chemist with the FDA’s Center for Biologics Evaluation and Research.

Science Watch correspondent Gary Taubes spoke with Beaucage from his FDA office in Bethesda, Maryland.

Let’s begin with the obvious. What is a nucleoside phosphoramidite and how does it work?

Beaucage: Generally speaking, one can describe a nucleoside phosphoramidite as a stable nucleic acid monomer that can be activated to a very reactive entity to enable rapid and efficient solid-phase DNA synthesis. Specifically, it's the basic tri-coordinated phosphorous function of the nucleoside phosphoramidite, which, upon activation with a weak acid, leads very quickly to complete oligonucleotide chain extension. The newly formed link is then chemically converted to the native tetra-coordinated state of the phosphate group found in both DNA and RNA. While the role of the nucleoside phosphoramidites in the synthesis of oligonucleotides appears simplistic, there is nonetheless a series of chemical steps involved in the process.

How did you get started on it?

Beaucage: In the late 1970s, Marv Caruthers was interested in studying specific protein-DNA interactions at the molecular level. He was asking his graduate students and post-docs to synthesize modified and unmodified segments of DNA, which were located near and at the expected protein-binding site, in order to precisely identify the functional groups on DNA that were recognized by the protein of interest.

But you had already been thinking for some time about a better way to synthesize oligos?

Beaucage: Yes. It was when, as a graduate student at McGill, I was using a two-step phosphotriester approach to synthesize oligonucleotides. The technique was so tedious and time-consuming that I could not help thinking about a better way to do this work. One day, when I was attending a graduate class on organic reaction mechanisms taught at the time by John T. Edwards, I became fascinated with the Hofmann degradation, which is a method whereby a stable amine function becomes a leaving group upon alkylation to a quaternary ammonium group. This reaction taught me that a stable entity could be activated to a reactive one when needed for specific purpose. Such a concept stayed in my mind until graduation and my moving to Boulder. Soon after my arrival in Caruthers's lab, and influenced by the recent findings of Letsinger and van Tamelen on the use of the phosphite coupling method for DNA and RNA oligonucleotide synthesis, I began searching the scientific literature for stable tri-coordinated organophosphorus compounds covalently linked to an amine group, and for ways to activate this amine function through quaternization or other means. I became aware of German and Russian reports describing the reaction of simple phosphoramidites with, for example, acids or alcohols. While these reports were inspiring—especially those involving reactions with amine salts—there were no indications suggesting that these phosphoramidites could be used to synthesize oligonucleotides. I thought it might be worthwhile to attempt the preparation of deoxyribonucleoside phosphoramidites and initiate a search for weak acids that might convert these phosphoramidites to reactive intermediates. That’s how it all began.

How quickly did it come together?

Beaucage: For the first year and a half of my post-doctoral stay nothing was really working, and I was having a hard time characterizing deoxyribonucleoside phosphoramidites. However, I came to realize that analyzing these phosphoramidites on silica gel thin layer chromatography plates was problematic. I discovered that silica gel was acidic enough to activate the phosphoramidites and induce decomposition of these species, presumably through hydrolysis. The positive outcome that stemmed from weeks of frustration was knowing that deoxyribonucleoside phosphoramidites could indeed be efficiently activated by weak acids and readily react with adventitious moisture. I then decided to monitor the preparation of deoxyribonucleoside phosphoramidites by phosporus-31 NMR. The Truth came out and everything became clear. I was able to evaluate the purity of phosphoramidites, study the activation mechanism of these compounds with various weak acids, and determine the kinetics and coupling efficiency of activated deoxyribonucleoside phosphoramidites directly in the NMR tube. Within the last six months of my NRCC postdoctoral fellowship, I knew almost everything about deoxyribonucleoside phosphoramidites and the application of this chemistry to solid-phase DNA synthesis. I was, nonetheless, aware that the stability of deoxyribonucleoside phosphoramidites in solution was not optimal for automated oligonucleotide synthesis. A discussion with Lincoln McBride, who was assigned to improve the emerging chemistry upon my departure from Caruthers's lab in 1981, led to structural refinement of the original phosphoramidites, which allowed facile purification of these compounds and resulted in adequate stability of the purified amidites in solution. An additional refinement to the phosphoramidite chemistry was later reported by Nanda Sinha in Hubert Koster's group on 1984. Since then, the phosphoramidite approach to solid-phase oligonucleotide synthesis has been essentially unchanged.

Why publish it in Tetrahedron Letters, instead of a more prestigious journal?