The effectivity of 1H-triazoles and -tetrazoles as activators in acid-catalyzed phosphoramidite alcoholysis

Article abstract of DOI:10.1002/hlca.200390157

The efficiency of 5-nitro-1,2,4-1H-triazole (3), 5-(methylthio)-1H-tetrazole (4), and 5-(4-nitrophenyl)-1H-tetrazole (5) as activators in phosphoramidite alcoholysis has been studied relative to 1H-tetrazole (6). Reactions of these azoles with diisopropyl (diisopropylamido)phosphite (1a) were followed in THF, and the rates were found to increase with increasing acidity of the azoles. The Bronsted a value of 0.7 determined for this dependence is in agreement with data published earlier.

Full text of DOI:10.1002/hlca.200390157

Helvetica Chimica Acta Vol. 86 (2003)
2005
The Effectivity of 1H-Triazoles and -Tetrazoles as Activators in
Acid-Catalyzed Phosphoramidite Alcoholysis
by Erkki. J. Nurminen*a), Jorma K. Mattinenb), and Harri Lˆnnberga)
a) Department of Chemistry, University of Turku, 20014 Turun yliopisto, Finland (phone: ‡358-(0)2-3336771;
fax: ‡358-(0)2-3336700, e-mail: enurmine@utu.fi)
b) Department of Organic Chemistry, äbo Akademi University, 20500 äbo, Finland
The efficiency of 5-nitro-1,2,4-1H-triazole (3), 5-(methylthio)-1H-tetrazole (4), and 5-(4-nitrophenyl)-1H-
tetrazole (5) as activators in phosphoramidite alcoholysis has been studied relative to 1H-tetrazole (6).
Reactions of these azoles with diisopropyl (diisopropylamido)phosphite (1a) were followed in THF, and the
rates were found to increase with increasing acidity of the azoles. The Br˘nsted a value of 0.7 determined for
this dependence is in agreement with data published earlier.
Introduction. Phosphoramidite alcoholysis is a reaction used in the coupling step
in automated oligonucleotide synthesis: a nucleoside 3'-phosphoramidite (1b) is
allowed to react repeatedly with the 5'-OH function of the growing, support-bound
oligonucleotide chain (Scheme 1) [1][2].
Scheme 1. Oligonucleotide Synthesis by the Phosphoramidite Approach
An acidic activator, commonly 1H-tetrazole (2), is used to accelerate the reaction.
During the last few years, demand for improvement of the already successful method
has increased, together with its application to large-scale synthesis of oligonucleotides.
Research on the mechanism of the reaction has been carried out, and new catalysts
have been studied as alternatives to the presently applied 1H-tetrazole [3]. Among
these candidates were more-acidic azole derivatives such as 5-(ethylthio)-1H-tetrazole
[4] and 5-(4-nitrophenyl)-1H-tetrazole [5].
This work is aimed at quantifying the relationship between the acidity of azoles and
their catalytic efficiency.
The tetrazole-promoted reactions of diisopropyl (diisopropylamido)phosphite (1a)
are known to be practically instant in MeCN [6]. In THF, however, the reaction kinetics
are slow and can be followed by 31P-NMR. The reaction is initiated by displacement of
the diisopropylamido group of 1a by the azole activator. The tetrazolide obtained is

2006
Helvetica Chimica Acta Vol. 86 (2003)
then rapidly converted to a phosphite triester. In fact, the tetrazole-activated
alcoholysis is almost as fast as the reaction of 1a with tetrazole [7].
Results and Discussion. In the present work, the reaction rates of 1a (0.1m) with 5-
nitro-1,2,4-1H-triazole (3), 5-(methylthio)-1H-tetrazole (4), and 5-(4-nitrophenyl)-
1H-tetrazole (5) were determined in THF at various activator concentrations (0.08
0.5m).
Scheme 2
The initial reaction rate was determined by fitting an exponential curve to the
observed time-dependent concentration of 1a (Fig. 1). The catalytic rate constant, kc,
was calculated for each azole according to Eqn. 1.
~
&
*
Fig. 1. Time-dependent concentrations of 1a during its reaction with 3 (0.19 m; ), 2 (0.17 m; ), 4 (0.18 m; ),
^
and 5 (0.19 m; ). Concentrations of 1H-tetrazole (2) were taken from [7] and normalized to fit those of the
other compounds.
ꢀ
ꢁ
d‰1aŠ
n
0
u0 ˆ lim
ˆ kc‰1aŠ ‰azoleŠ
(1)
0
t!0
dt
The reaction order in azole concentration was invariably found to be higher than
unity, the nonlinear character increasing together with the pKa value of the activator

Helvetica Chimica Acta Vol. 86 (2003)
2007
(Table and Fig. 2). However, for all the activators, a reasonable fit (r> 0.97) was
obtained when using a second-order dependence of the rate on the azole concentration.
This allowed us to quantitatively compare the efficiency of 3 5 with that of 1H-
tetrazole (2) studied earlier [7].
~
&
*
Fig. 2. Nonlinear dependence of v0/[1a] and activator concentration in the reactions of 1a with 3 ( ), 6 ( ), 4 ),
^
and 5 ( ), respectively
Table. Reaction of Azole Activators with 1a
Compound
pKa
n (best fit)
kc/mÀ3 sÀ1
5-(4-Nitrophenyl)-1H-tetrazole (5)
5-(Methylthio)-1H-tetrazole (4)
1H-Tetrazole (2)
3.66
4.15
4.87
5.19
1.4
1.5
2.2
2.9
2.71
0.76
0.31
0.19
5-Nitro-1,2,4-1H-triazole (3)
When the calculated kc values were plotted logarithmically against the respective
pKa values in THF [8], a Br˘nsted a value of 0.7 was obtained (Fig. 3). This is in
‡
agreement with the reaction being acid-catalyzed: for the H4N ClÀ-catalyzed reaction,
a values of 0.05 0.65 have been determined in neat alcohol [9], while ammonium
trifluoromethanesulfonate, methanesulfonate, and trifluoroacetate gave rise to values
of 0.6 0.9 in MeCN [6].
According to kinetic studies, 1H-tetrazole and other acidic azoles also act as
nucleophilic activators in the phosphoramidite alcoholysis that proceeds via a reactive
azolidite intermediate [7]. The reaction rate has been observed to depend on the
nucleophilicity of the activator. For ammonium salts in MeCN, a bnucl value of 0.2 has
been reported, given the pKa of an activator is used as a measure of its nucleophilicity
[6]. This means that more-acidic azoles (better acid catalysts) are inferior to less-acidic
ones regarding the nucleophilic contribution of the activation. Consequently, the a
value of 0.7 determined in this work may actually refer to an even more complete
proton transfer in the transition state, but the rate-accelerating effect of the increased
acidity of the activator is partially cancelled by the lower nucleophilicity of its conjugate
base.

2008
Helvetica Chimica Acta Vol. 86 (2003)
Fig. 3. Br˘nsted plot for the reaction of 1a with the activators 3 6 performed in THF at 208
Experimental Part
Diisopropyl (diisopropylamido)phosphite (1a) was prepared as described in [7]. THF was dried over CaH2.
The azole activators 3 6 were used as purchased, and their THF solns. were dried over molecular sieves (4 ä).
In the kinetic studies, the reagents were mixed in a septum-sealed NMR tube, and the reactions were followed
by 31P-NMR spectroscopy (202 MHz) as described in [7]. The level of the hydrolysis reaction was kept lower
than 7.5% in all kinetic runs.
REFERENCES
[1] S. L. Beaucage, R. P. Iyer, Tetrahedron 1992, 48, 2223.
[2] E. E. Nifant×ev, M. K. Grachev, Russ. Chem. Rev. 1994, 63, 575; E. E. Nifant×ev, M. K. Grachev, S. Yu,
Burmistrov, Chem. Rev. 2000, 100, 3755.
[3] E. J. Nurminen, H. Lˆnnberg, Eur. J. Org. Chem. 2002, submitted.
[4] P. Wright, D. Lloyd, W. Rapp, A. Andrus, Tetrahedron Lett. 1993, 34, 3373; R. Vinayak, F. Colonna, D.
Tsou, B. Mullah, A. Andrus, B. Sproat, Nucleic Acids Symp. Ser. 1994, 31, 165.
[5] B. C. Froehler, M. D. Matteucci, Tetrahedron Lett. 1983, 24, 3171; A. Wolter, J. Biernat, H. Kˆster,
Nucleosides Nucleotides 1986, 5, 65; R. T. Pon, Tetrahedron Lett. 1987, 28, 3643.
[6] E. J. Nurminen, J. K. Mattinen, H. Lˆnnberg, J. Chem. Soc., Perkin. Trans. 2 2001, 2159.
[7] E. J. Nurminen, J. K. Mattinen, H. Lˆnnberg, J. Chem. Soc., Perkin. Trans 2 1998, 1621.
[8] E. J. Nurminen, J. K. Mattinen, H. Lˆnnberg, J. Chem. Soc., Perkin. Trans. 2 1999, 2551.
[9] E. E. Nifant×ev, M. K. Grachev, S. Yu. Burmistrov, L. K. Vasyanina, Dokl. Chem. (Engl. Transl.) 1988, 303,
320; E. E. Nifant×ev, M. K. Gratchev, S. Yu. Burmistrov, L. K. Vasyanina, M. Yu. Antipin. Yu. T. Struchkov,
Tetrahedron 1991, 47, 9839; S. Yu. Burmistrov, N. M. Shchedrova, M. K. Grachev, L. K. Vasyanina, E. E.
Nifant×ev, Russ. J. Gen. Chem. (Engl. Transl.) 1995, 65, 500.
Received December 14, 2002