2,4-Dinitrophenol: A novel activating reagent in nucleotide synthesis via the phosphoramidite route. Design of new effective phosphitylating reagents

Article abstract of DOI:10.1016/S0040-4039(00)01178-3

2,4-Dinitrophenol (DNP) is a remarkably efficient activator in the reaction of P(III) amides with nucleosides to give P(III) esters in excellent yield. Typical examples of this novel procedure are presented herein. Mechanistic features of the activation were elucidated by model studies of the reaction of bis(2-cyanoethyl)diisopropylphosphoroamidite with benzyl alcohol. The importance of the initial protonation stage and formation of an intermediate P(III)-2,4-dinitrophenyl ester were clearly demonstrated by ³¹P NMR spectroscopy and independent synthesis. New phosphitylating reagents containing the 2,4-dinitrophenoxy group do not require any activation. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01178-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 7535–7539
2,4-Dinitrophenol: a novel activating reagent in nucleotide
synthesis via the phosphoramidite route. Design of new
effective phosphitylating reagents†
Wojciech Da˛bkowski,a Izabela Tworowska,a Jan Michalskia,* and Friedrich Cramerb
aCentre of Molecular and Macromolecular Studies, Polish Academy of Sciences, 90-363 L*o´dz´,
Sienkiewicza 112, Poland
bMax-Planck-Institut fu¨r experimentelle Medizin, Hermann-Rein-Straße 3, 3400 Go¨ttingen, Germany
Received 12 April 2000; revised 30 June 2000; accepted 12 July 2000
Abstract
2,4-Dinitrophenol (DNP) is a remarkably efficient activator in the reaction of P(III) amides with
nucleosides to give P(III) esters in excellent yield. Typical examples of this novel procedure are presented
herein. Mechanistic features of the activation were elucidated by model studies of the reaction of
bis(2-cyanoethyl)diisopropylphosphoroamidite with benzyl alcohol. The importance of the initial protona-
tion stage and formation of an intermediate P(III)-2,4-dinitrophenyl ester were clearly demonstrated by
31P NMR spectroscopy and independent synthesis. New phosphitylating reagents containing the 2,4-dini-
trophenoxy group do not require any activation. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: nucleosides; nucleotides; phosphoroamidites; 2,4-dinitrophenol; synthesis.
Phosphoroamidites are among the most important reagents in the synthesis of phosphates of
biological interest by the ‘phosphite approach’. Tetrazole, tetrazole salts with amines and
structural analogues of this heterocycle are generally used as activators for the reaction between
alcohols and phosphoramidites. Tetrazole, the most widely employed activator, must often be
used in excess and be of high purity, which involves hazardous sublimation.1 Other activators
like amine hydrochlorides and acyl chlorides have been used less frequently.2 In spite of
impressive progress in this field there are still important problems to be solved. When the
necessity arises to produce biophosphates in large quantities, better procedures are required for
activation of phosphoramidites.
In our studies on the synthesis of modified nucleosides we faced the challenge of finding an
alternative mode of activation which did not use tetrazole or similar compounds. Recently we
have found that trimethylchlorosilane, Me3SiCl, is a remarkably good activator in the reaction
of phosphoroamidites with nucleosides.3
* Corresponding author. Fax: (042) 6847126; e-mail: jmich@bilbo.cbmm.lodz.pl
† This paper is dedicated to the memory of Professor Alexander Krayevsky.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01178-3

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In a search for other effective ‘non-classical’ activators we turned our attention to strongly
acidic phenols. We have found that 2,4-dinitrophenol (DNP), which with a pKa value of 4.1 is
similar to that of tetrazole (pKa=4.9), acts as an efficient activator of phosphate synthesis via
the phosphoroamidite procedure. The reaction of P(III) amidites with an equivalent amount of
nucleoside in the presence of DNP proceeds in very high yield and at rates comparable or higher
than those when tetrazole is used. Phosphitylations activated by DNP take place at room
temperature in solvents like THF, CH2Cl2 or MeCN. On average the amount of activator
required for efficient coupling is ca. 1.5 equivalents of the stoichiometrical ratio.4 Most of our
experiments were performed with P(III) amides derived from diisopropylamine in order to
conform with the most popular phosphitylation procedures. Selected examples illustrating our
methodology are chosen from nucleotide chemistry. DNP is distinctly superior to tetrazole when
P(III) amides bear a strongly electron-attracting group at the phosphorus centre.5
The coupling of amidite 1 prepared according to Tataku et al.6 with 5%-DMTr-thymidine 2
proceeds in a chemoselective way according to Scheme 1 to give the amidite 3 (98% yield as
determined by 31P NMR). In this and all other cases, no removal of DMTr and Pix
(9-(9-phenyl)xanthenyl) protective groups is observed.
Scheme 1.
The amidite 3 when allowed to react with 3%-pixyl-thymidine 4 gives the dinucleoside
phosphite 5 (95% yield as determined by 31P NMR) (Scheme 2).
Scheme 2.

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The fluoroamidite 6 prepared in our earlier studies7 reacts with 3%-DMTr-thymidine 5 in the
presence of DNP yielding the dinucleoside phosphorofluoridite 7 in excellent yield (Scheme 3).
This compound is identical with samples prepared by us with the aid of other activators.7
Scheme 3.
When pure diastereoisomers of 6 were used8 the coupling of Scheme 3 proceeds in a
non-stereoselective way giving a (1:1) mixture of the corresponding diastereoisomers of the
fluoroamidites 7 (98% yield as determined by 31P NMR).
The mechanism of activation by DNP is presumed to involve its reaction with P(III) amidite
in a similar manner to that accepted for activation in the presence of tetrazole.9 We anticipated
formation of the intermediate bearing the 2,4-dinitrophenoxy group attached to the P(III)
center. This assumption was confirmed by a model experiment involving the reaction of
bis(2-cyanoethyl)-N,N-diisopropylphosphoroamidite 8 and benzyl alcohol in the presence of 1.5
equivalents of DNP. Formation of the intermediate 9 (31P=140.1 in C6D6) en route to the
formation of benzyl-bis-(2-cyanoethyl)phosphite 10 was observed by 31P NMR spectroscopy. In
order to verify the structure of the intermediate 9, its independent synthesis was carried out
according to Scheme 4 from the chloridite 11 and trimethylsilyl 2,4-dinitrophenol 12. The
phosphite 9 prepared in this way reacts spontaneously with benzyl alcohol to give the phosphite
10 (97% yield as determined by 31P NMR).
PhCH2OH
+
(RO)2P OAr
9
(RO)2P OCH2Ph
10
(RO)2P Cl
11
Me3SiOAr
12
_
Me3SiCl
31
31
P NMR: 148.9
P NMR: 140.1
RO = OCH CH CN, ArO = 2,4-dinitrophenoxy
2
2
Scheme 4.

7538
The mechanistic features of the DNP activation are shown in Scheme 5.
H
RO
RO
RO
RO
P
+
ArOH
P
ArO
+
N
fast
N
8
ArOH
slow
RO
RO
RO
RO
HOCH2Ph
ArOH
P
OCH2Ph
H2N
P
OAr
+
-
10
9
RO = OCH CH CN, ArO = 2,4-dinitrophenoxy
2
2
Scheme 5.
The signal intensity of 9 increases with increasing amount of DNP. Further evidence for the
mechanism described in Scheme 5 is that addition of triethylamine dramatically reduces the rate
of formation of the phosphite 10. This inhibition provides evidence for reversible acid catalysis.
The nucleophilic catalysis step is slow and cannot explain the observed inhibition. The practical
consequence of this study is that DNP must be used in at least an equimolar amount. The fact
that the reaction described in Scheme 3 proceeds in a non-stereoselective way is explained by an
epimerization at the chiral phosphorus atom due to exchange of ligands with the excess DNP
present in the reaction medium. This situation is similar to that observed for the activation of
phosphoramidates by tetrazole. In conclusion, two important factors are involved in catalytic
properties of DNP: acidity and the ability to form reactive intermediates.
A useful consequence of these studies is the possibility of designing new phosphitylating
reagents which react spontaneously with alcohols without any activation. For example bis(2,4-
dinitrophenyl)phosphoramidite 13 was prepared by a standard procedure from the correspond-
ing aminodichlorophosphine. Amide 13 reacts with nucleosides in a non-selective way and a
mixture of products is formed. From a mechanistic point of view this result is consistent with
spontaneous displacement of a 2,4-dinitrophenoxy group which liberates free DNP. The latter
activates the amido group leading to further ligand exchange. However, if the phosphitylation
by 13 is performed in the presence of one mole of triethylamine, high chemoselectivity is
observed, as shown in Scheme 6. The amidate 14 is formed in good yield (92% yield as
determined by 31P NMR).
DNP proved to be an excellent activator for the hydrolysis of phosphoroamidates to yield the
corresponding hydrogen phosphonates. For example: from the amidite 3 the corresponding
H-phosphonate diester was obtained in very high yield as a result of hydrolytic replacement of
the amido group.

7539
Scheme 6.
Acknowledgements
This work was supported by the Stiftung fu¨r Deutsch-Polnische Zusammenarbeit (2789/96/
LN) and the German–Polish project (POL-211-96).
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