Scientific Profile: Fritz Eckstein, PhD – A summary of a lifetime of work
Eckstein Science with reference to oligonucleotides
The development of oligonucleotides for therapy rests to a certain extent on their chemical characteristics. Eckstein’s work has contributed two such modifications which have found introduction in many of these oligonucleotides. These are the phosphorothioates and the 2’-fluoro substituent, the latter in RNA oligonucleotides. Here is a brief description of how these modifications were part of Eckstein’s research.
After entering the area of nucleic acids in 1964 at the Max-Planck-Institute in Goettingen and after some not so successful efforts to develop the formation of the internucleotidic linkage, he began to change the phosphate to a phosphorothioate for no particular purpose but just to see whether this small change would, if at all, influence the interaction with enzymes. Adenosine 5’- phosphorothioate was prepared and incubated with alkaline phosphatase in 1970. Quite surprisingly there was no reaction, the compound in contrast to adenosine 5’-phopshate was stable. From a chemical point of view this was not anticipated as the exchange of an oxygen by a sulfur is a rather conservative one. An explanation for this resistance to degradation will be given later.
This surprising observation stimulated an extension to phosphorothioate diesters with the sulfur in a non-bridging position. One of the first was uridine 2’,3’-cyclic phosphorothioate. Here the phosphorus is chiral and as a consequence we have two diastereomers which of course is true for the internucleotidic phosphorothioate linkage such as in oligoucleotides but also for the nucleoside 5’-triphosphate analogs with the sulfur at the α or the ß position, such as in ATPαS or ATPßS. Only the Sp-isomer of the NTPαS is a substrate for DNA- and RNA-polymerases with inversion of configuration, resulting in the Rp-diastereomer of the internucleotidic linkage. The chemical synthesis of oligonucleotides, however, results in a mixture of the two isomers.
An oligonucleotide with a mixture of the isomers, which can be separated by chromatography, at the 3’-terminus has been investigated as a substrate for the Klenow fragment of DNA polymerase 1. The Rp isomer is a proper substrate whereas the Sp is not. The X-ray analyses of the three protein complexes, performed by Brautigam and Steitz and, with either the phosphate terminus or that with the Sp or the Rp isomers, provide a structural explanation for the resistance of the Sp isomer. Two metal ions are at the active site which are involved in catalysis. For the Sp isomer, one of the metal ions is been dislocated because of the somewhat larger van-der-Waals radius of the sulphur which prevents catalysis. Thus this is so far the only explanation for the lack of substrate properties by a phosphorothioate. It is the replacement of a group essential for catalysis. Even though this is demonstrated here for the displacement of a metal ion, one can easily imagine for other enzymes a protein functional group to be dislocated and thus preventing activity. Another explanation for the inactivity of the phosphorothioates in enzymatic reaction is that in the transition state, there is no place for the sulphur, but this has not been rigorously shown.
First in vivo data
The first experiments with polynucleotides containing phosphorothioates were conducted in 1968. Poly r(A-U) was prepared by DNA-dependent RNA polymerase using ATP and UTPαS providing the polynucleotide with the phosphorothioate Rp isomer at alternating positions. Incubation of the polynucleotide with snake venom phosphodiesterase showed considerable resistance to degradation in comparison to the unmodified polynucleotide. Had we been able to prepare the polynucleotide with the Sp isomer, the polynucleotide would have been essentially totally resistant. These results stimulated collaboration with Tom Merigan and Erik De Clercq at Stanford Medical School in 1969. They examined the induction of interferon with poly r(A-U) with the phosphorothioates throughout. The modified polynucleotide stimulated the interferon production 2-to 20-fold and the cellular resistance to viral infection 100- to 10,000-fold. Increase of interferon was also observed in the rabbit. This was presumed to be the result of high stability towards enzyme degradation of the modified polynucleotide as shown for pancreatic ribonuclease and enzymes present in calf serum. Again, these were only the Rp-isomers!
These data are the basis for the phosphorothioate introduction into therapeutic oligonucleotides. Later discovered properties are the difference of phosphate and phosphorothioate coodination to metal ions pointed out by Mildred Cohn, and very recently the improved uptake of the phosphorothioate oligonucleotides in vivo.
This modification was first introduced by chemical synthesis into the Hammerhead ribozyme in 1991. The modified ribozyme did not show any appreciable decrease in activity but had resistance to degradation by ribonuclease and rabbit serum. Thus, this modification exhibited desirable properties for RNA oligonucleotides. Later we showed that the 2’-modified nucleotides could be incorporated by T7 RNA polymerase facilitating their incorporation. These results made this modification a preferred one for RNA aptamers where it is incorporated frequently.
Thank you to Dr. Eckstein for providing the content for this article. He wishes to convey the following:
“The interaction with many colleagues over the years has been most fruitful and delightful. Their contributions have helped considerably to establish these chemistries in various aspects.”