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Beaucage S L & Caruthers M K. Deoxynucleoside phosphoramidites—a new class of
key intermediates for deoxypolynucleotide synthesis.
Tetrahedmn Lett. 22:1859-62, 1981.
[Department of Chemistry. University of Colorado. Boulder. CU]
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action With properly protected deoxynucleosides, eliminate the formation of undesired
3’-3’-dinucleoside monophosphites and afford
high yields of the deoxynucleoside phosphoramidites. Because these intermediates lack
functional groups susceptible tohydrolysis, we
also anticipated that their isolation and purification would be easier than with the deoxy.
Serge L Beaucage
nucleoside chlorophosphites. Initially, our
hypotheses were difficult to support because
Department of Genetics
preliminary results on silica gel thin-layer chroStanford University School of Medicine
matography appeared unsatisfactory. We raStanford, CA 94305
tiönalized that silica gel might havebeen acidic enough to activate the deoxynucleoside
phosphoramidites and generate inconsistent
January 18, 1987
data. ____________________
The use of 31p NMR was the crucial step in
the development of the deoxynucleosidephosphoramidite chemistry since it was free of
Upon graduation from McGill University in
1978, I joined Marvin Caruthers’s group at the
these problems. The 31p NMR data rapidly
University of Colorado as a postdoctoral fel- corroborated ourtheories. As anticipated, the
low. At that time, my project involved site- deoxynucleoside phosphoramidites were effidirected mutagenesis of the SV4O T-antigen
ciently prepared and subsequently isolated as
stable amorphous solids. These intermediates
binding sites. Synthetic DNA required for the
were also shown by 31p NMR to be easily
project was prepared using the phosphite cou2 activated by weak acids such as 1 H-tetrazole.
pling method of Letsinger and coworkers.’
Their approach, involvingthe use of deoxynu- When activated deoxynucleoside phosphoramidites were then added to a deoxynucleocleoside chlorophosphites as intermediates,
permitted unprecedented kinetics in the for- side attached covalently to a polymeric supmation ofintemucleotidic linkages, but despite port, condensation toform a dinucleotide was
its revolutionary features, the methodology complete in less than one minute. The efficacy
had limitations. These were the instability of of the deoxynucleoside phosphoramidites in
the deoxynucleotide chlorophosphite syn- solid-phase DNA synthesis became history.
thons, their high reactivity towards moisture
Our Tetrahedron Letters paper is highly
(which meant they had to be stored and han- cited probably
because, after further refine35
dled under inert gas), and the generation of ments, - the deoxynucleoside phosphorlarge amounts of unwanted 3’-3’-dinucleoside amidite methodology became “the chemistry
monophosphites. Such complications clearly that catapulted automated DNA synthesis into
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compromised automation of the procedure.
many laboratories” and co’ntributed, along
We reasoned that replacing a chlorine atom
with DNA sequencing and other basic develof the phosphorodichloridite by a dimethyl- opments in recombinant DNA technology, to
amino group would generate a monofunction- many recent discoveries in molecular biology
al phosphitylating agent that would, upon re- and genetics.
This paper describes the preparation and characterization of deoxynucleoside phosphoramidites. The
usefulness of these intermediates in solid-phase DNA
synthesis upon activation
5 by weak proton donors is
also presented. [The SC! indicates that this paper has
been cited in over 265 publications.]
I. Letsinger R L, Finnan J 1, Heavner C A & Lunsford W B. Phosphite coupling procedure for generating internucleotide
links. I. Amer. Chem. Soc. 97:3278-9. 1975. (Cited 50 times.)
2. Let.singer R L & Lunsford W B. Synthesis of thymidine otigonucleotides by phosphite nester intermediates.
I. Amer. Chem. Soc. 98:3655-61. 1976. (Cited t25 times.)
3. McBride L J & Caruthers M H. An investigation of several deonynucleoside phosphoramidites useful for synthesizing
deoxyoligonucleotides. Teuahcdmn Left. 24:245-8. 1983.
4. Adams S P. Kavka K S. Wykes E J, Holder S B & Galluppi C R. Hindered dialkylamino nucleoside phosphite reagents
in the synthesis of reo DNA 51-mers. I. Amer. Chem. Soc. 105:661-3, 1983.
5. Sinha N D, Bjernal J, McManus J & Kts*er H. Polymer support oligonucleotidc synthesis. XVIII: use of fl-cyanoethylN.N-dialkylamino.N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA frugments simpli~’ing
deprotection and isolation of the final product. Nun. Acid. Res. 12:4539-57, 1984.
6. Kaplan B E. The automated synthesis of oligodeonynucleotides. Trends Biotech. 3:253-6. 1985.
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