Solution stability and degradation pathway of deoxyribonucleoside phosphoramidites in acetonitrile

Article abstract of DOI:10.1081/NCN-120039215

The impuritiy profiles of acetonitrile solutions of the four standard O-cyanoethyl-N,N-diisopropyl-phosphoramidites of 5′-O-dimethoxytrityl (DMT) protected deoxyribonucleosides (dG^ib, dA^bz, dC ^bz, T) were analyzed by HPLC-

Full text of DOI:10.1081/NCN-120039215

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Solution Stability and Degradation Pathway
of Deoxyribonucleoside Phosphoramidites in
Acetonitrile
Achim H. Krotz Ph.D. a , Claus Rentel a , Dennis Gorman a , Phil Olsen a , Hans J. Gaus a
James V. McArdle a & Anthony N. Scozzari a
,
a Drug Substance Manufacturing, Isis Pharmaceuticals, Inc., 2292 Faraday Ave., Carlsbad,
California, 92008, USA
Published online: 17 Aug 2006.
To cite this article: Achim H. Krotz Ph.D. , Claus Rentel , Dennis Gorman , Phil Olsen , Hans J. Gaus , James V. McArdle
& Anthony N. Scozzari (2004) Solution Stability and Degradation Pathway of Deoxyribonucleoside Phosphoramidites in
Acetonitrile, Nucleosides, Nucleotides and Nucleic Acids, 23:5, 767-775, DOI: 10.1081/NCN-120039215
To link to this article: http://dx.doi.org/10.1081/NCN-120039215
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, No. 5, pp. 767–775, 2004
Solution Stability and Degradation Pathway of
Deoxyribonucleoside Phosphoramidites
in Acetonitrile
*
Achim H. Krotz, Claus Rentel, Dennis Gorman, Phil Olsen,
Hans J. Gaus, James V. McArdle, and Anthony N. Scozzari
Isis Pharmaceuticals, Inc., Carlsbad, California, USA
ABSTRACT
The impuritiy profiles of acetonitrile solutions of the four standard O-cyanoethyl-N,N-
diisopropyl-phosphoramidites of 5’-O-dimethoxytrityl (DMT) protected deoxyribo-
nucleosides (dGib, dAbz, dCbz, T) were analyzed by HPLC-MS. The solution stability
of the phosphoramidites decreases in the order T, dC > dA > dG. After five weeks
storage under inert gas atmosphere the amidite purity was reduced by 2% (T, dC), 6%
(dA), and 39% (dG), respectively. The main degradation pathways involve hydrolysis,
elimination of acrylonitrile and autocatalytic acrylonitrile-induced formation of
cyanoethyl phosphonoamidates. Consequently, the rate of degradation is reduced by
reducing the water concentration in solution with molecular sieves and by lowering
*
Correspondence: Achim H. Krotz, Ph.D., Drug Substance Manufacturing, Isis Pharmaceuticals,
Inc., 2292 Faraday Ave., Carlsbad, CA 92008, USA; Fax: (760) 603-4655 ; E-mail: akrotz@
isisph.com.
767
DOI: 10.1081/NCN-120039215
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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Krotz et al.
the amidite concentration. Acid-catalyzed hydrolysis could also be reduced by
addition of small amounts of base.
Key Words: Degradation pathway; Deoxyribonucleoside; Phosphoramidite;
Stability.
INTRODUCTION
Oligodeoxyribonucleotides are synthesized through phosphoramidite chemistry on a
polymeric support by sequential addition of nucleoside building blocks [O-Cyanoethyl-
N,N-diisopropylphosphoramidites of protected nucleosides: dAbz (1a), dCbz (1b), dGib
(1c), T (1d), bz = benzoyl, ib = isobutyryl] to a growing oligonucleotide chain which is
covalently attached to the solid support (Scheme 1).[1–3] Prior to solid-phase synthesis
the phosphoramidites are dissolved in anhydrous acetonitrile at concentrations of ca 0.2
M. Frequently, reagent solutions are prepared for multiple syntheses and used over a
period of days. Due to the increasing demand for both large scale production and high
throughput synthesis of oligonucleotides we investigated the stability of 1a–d after
dissolution in acetonitrile. In this report, we describe the formation of the major
degradation products and provide a mechanistic rationale.
RESULTS AND DISCUSSION
O-Cyanoethyl-N,N-diisopropyl-phosphoramidites 1a–d are starting materials in the
synthesis of oligonucleotides and phosphorothioate oligodeoxyribonucleotides, a
promising new class of therapeutic agents used to treat various diseases through
antisense mechanisms of action.[4] The 5’-O protecting group is 4,4’-dimethoxytrityl
(DMT), the protecting group for the exocyclic amino groups of adenine and cytosine
are benzoyl and for guanine it is isobutyryl. Upon activation with a weak organic
acid (1H-tetrazole or an alternative activating agent) phosphoramidites 1 couple to
the 5’-hydroxyl function of a support-bound oligonucleotide.[5] To obtain high
coupling efficiency throughout the synthesis, a molar excess of 1 is used. Typical
synthesizer programs use the same volume of phosphoramidite solution for each
coupling. Ideally, oligonucleotide synthesis is performed with a minimum molar
excess of reagent at each coupling. Further reduction of the amidite excess through
Scheme 1. Chemical structure of deoxyribonucleoside phosphoramidites.

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Table 1. Analytical characterization of compounds 2a-d and 4a-d.
769
Mass
(calcd.)
Compound
Formula
Mass (found)a
797.4 [M + Na]+
31P NMRb
2a
2b
2c
2d
4a
4b
4c
C41H39N6O8P
C40H39N4O9P
C38H41N6O9P
C34H36N3O9P
C47H52N7O7P
C46H52N5O8P
C44H54N7O8P
774.3
750.3
756.3
661.2
857.4
833.4
839.4
8.5, 8.6
8.6, 8.7
8.7, 8.9
773.3 [M + Na]+, 749.1 [M À H]À
757.3 [M + H]+, 779.3 [M + Na]+
684.3 [M + Na]+, 660.1 [M À H]À
880.4 [M + Na]+, 856.3 [M À H]À
856.4 [M + Na]+, 832.4 [M À H]À
840.5 [M + H]+, 862.4 [M + Na]+,
838.3 [M À H]À
8.6, 8.7
32.0, 32.3
32.3–32.9
32.3–33.7
4d
C40H49N4O8P
744.3
767.4 [M + Na]+, 743.3 [M À H]À
32.2–32.6
aMass spectra were obtained in positive and/or negative mode.
bsolvent: perdeuteroacetonitrile.
2a–d, see Refs. [6-8].
3a–d were purchased from Glen Research, Sterling, Virginia.
Preparation of 4a–d. A solution of protected nucleoside phosphoramidite (DMT-dAbz, DMT-dCbz,
DMT-dGib, DMT-T, 10 g), acrylonitrile (5 ml) and acetonitrile (50 ml) was heated at 60°C for 24 to
36 h under exclusion of moisture. The solution was evaporated and the residue was purified by
flash-chromatography on silica using a gradient (ethyl acetate/0.1% triethylamine to ethyl acetate/
methanol (9:1, v/v)/0.1% triethylamine. Yield 50 to 90%.
degradation of the phosphoramidite would lead to lower coupling efficiency resulting
in an overall reduction of yield. Also, consideration needs to be given to the
structure of the degradation products with respect to reactivity and potential impact
on oligonucleotide quality.
Homogenous solutions of 1a–d (0.2 M) in anhydrous acetonitrile (water content
< 30 ppm) were prepared. To exclude absorption of moisture from the air the solutions
were stored under a blanket of argon in stainless steel containers. Samples were
removed at different time points and analyzed by HPLC/MS. The identity of the
degradation products was confirmed by comparison of retention times and mass spectral
data (molecular weight, fragmentation) with authentic reference material (Table 1).[6–8]
A summary of structure assignments and quantitation of the degradation products
by HPLC-UV (relative area-% at wavelength 260 nm) of the individual phosphor-
amidites is shown in Table 2. A representative RP-HPLC profile is shown in Fig. 1.
The relative amount of intact phosphoramidite in solution as function of time is shown
in Fig. 2. The proposed degradation pathway is shown in Scheme 2.
Phosphoramidites used in this study had initial HPLC UV-purities between 98.8
and 99.6 area-% (at wavelength 260 nm). Degradation of dG amidite 1c proceeds
significantly faster than degradation of dA, dC or T amidite. The rate of degradation
decreases in the order dG > dA > dC ꢀ T (Fig. 2). After 10 days the initial purity of
dG amidite has been reduced by 7.5%, the main degradation products being CE-H-
phosphonate (2), H-phosphonate (3) and CE-phosphonoamidate (4), combined
accounting for 98% of the degradation products (Table 2). For comparison, over
the same period of time the purity of dA, dC and T amidite was reduced only by

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Krotz et al.
Table 2. (a) Composition of dA phosphoramidite 1a solution as function of time (area-%
by HPLC), (b) Composition of dC phosphoramidite 1b solution as function of time (area-% by
HPLC), (c) Composition of dG phosphoramidite 1c solution as function of time (area-% by HPLC),
(d) Composition of T phosphoramidite 1d solution as function of time (area-% by HPLC).
Compound
Initial
Day 4
Day 7
Day 10
Day 18
Day 35
a
dA amidite (1a)
99.58
0.03
n.d.
99.18
0.18
0.03
0.06
0.55
99.01
0.27
0.05
0.09
0.58
98.87
0.35
0.12
0.11
0.55
98.17
0.58
0.30
0.17
0.78
93.56
2.39
2.58
0.86
0.61
CE-H-phosphonate (2a)
H-phosphonate (3a)
CE-phosphonoamidate (4a)
Sum of other impurities
b
n.d.
0.39
dC amidite (1b)
98.91
0.11
98.71
0.27
0.01
n.d.
98.68
0.32
0.02
n.d.
98.50
0.39
0.04
n.d.
98.38
0.51
0.11
0.04
0.96
96.90
0.56
0.66
0.13
1.75
CE-H-phosphonate (2b)
H-phosphonate (3b)
CE-phosphonoamidate (4b)
Sum of other impurities
c
< 0.01
n.d.
0.97
1.01
0.98
1.07
dG amidite (1c)
99.02
0.19
97.30
0.93
0.75
0.21
0.81
95.39
1.57
1.68
0.42
0.94
91.53
2.54
3.63
1.16
1.14
81.49
2.38
5.50
5.27
5.36
59.96
1.53
8.08
CE-H-phosphonate (2c)
H-phosphonate (3c)
CE-phosphonoamidate (4c)
Sum of other impurities
d
< 0.01
0.07
0.71
19.96
10.47
T amidite (1d)
98.79
0.15
98.66
0.27
0.02
0.04
1.01
98.6
0.31
0.02
0.04
1.03
98.38
0.36
0.03
0.04
1.19
98.48
0.51
0.09
0.04
0.88
97.23
1.17
0.51
0.04
1.05
CE-H-phosphonate (2d)
H-phosphonate (3d)
CE-phosphonoamidate (4d)
Sum of other impurities
< 0.01
0.05
1.00
n. d.: none detected.
0.7%, 0.4% and 0.4%, respectively. A common degradation pathway shown in
Scheme 2 is found for the four phosphoramidites.
Formation of the Major Degradation Products of dG Amidite
In the context of oligonucleotide synthesis, the solution stability of 1a, 1b and 1d
in anhydrous acetonitrile is acceptable. Degradation of less than 1% starting material
over a period of ten days will have little to no effect on the outcome of oligonucleotide
synthesis. Hence, we focused our investigation on dG phosphoramidite 1c. With
degradation products being essentially identical in all four phosphoramidites the
conclusions drawn here may be applied to the other phosphoramidites. Based on
impurity profile analysis of phosphoramidite solutions stored for up to 35 days the
following degradation pathway is evident. (Scheme 2) Degradation is initiated by the
acid-catalyzed hydrolysis reaction of phosphoramidite 1 with water (water serves as its
own activator) leading to formation of CE-H-phosphonate 2 and diisopropylamine.
Figure 3A shows the initial rapid formation of degradation product 2 (2.5% after

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771
Figure 1. Representative HPLC profiles of amidite 1c (Table 3, entry 1, lower trace) showing the
formation of CE-H-phosphonate 2c, H-phosphonate 3c and CE-phosphonoamidate 4c, respectively
(Table 3, entry 2, upper trace) after 10 days of storage as solution in acetonitrile.
10 days). The water content of the individual phosphoramidites was as follows: dA
(0.23%, w/w), dC (0.12%, w/w), dG (0.11%, w/w) and T (0.19%, w/w). Due to the ca
40- to 50-fold difference in molecular weight between phosphoramidites 1 and water, a
water content of 0.1 to 0.2%, w/w, may cause 5 to 10% hydrolysis either upon storage
or during oligonucleotide synthesis upon activation prior to coupling. Despite having
the lowest water content 1c degrades fastest, indicative of its intrinsic high reactivity at
the phosphoramidite functionality.
After 10 days the water content is largely reduced and also the concentration of
the liberated base (diisopropylamine) is high enough (ca 6 mol-%) to slow down the
Figure 2. Stability of phosphoramidites 1a–d in acetonitrile solution (0.2 M) at room
temperature. (View this art in color at www.dekker.com.)

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Krotz et al.
Scheme 2. Degradation pathway of deoxyribonucleoside phosphoramidites.
acid-catalyzed hydrolysis reaction (see below). The presence of base leads to
elimination of acrylonitrile of CE-H-phosphonate 2. Comparing Fig. 3A and 3B it
becomes apparent that the maximum rate of formation of H-phosphonate 3 is near day
10, when the concentration of CE-H-phosphonate 2 is near the maximum. Concurrent
with the formation of 3 is the formation of acrylonitrile. Reaction of acrylonitrile with
phosphoramidite 1 leads to formation of phosphonoamidate 4 (Fig. 3C). In this
reaction, acrylonitrile is both a reagent and a reaction product. Therefore, once started,
this reaction continues until the phosphoramidite is consumed making 4c the major
degradation product. The amount and concentration of acrylonitrile present is
determined by the extent of the initial hydrolysis reaction. One may therefore expect
to decrease the rate of phosphoramidite degradation by slowing down the rate of
hydrolysis. This can be accomplished by reducing the concentration of the reactants
(phosphoramidite, water) or by the addition of an inhibitor (a base) in order to reduce
the acidity of the medium. (Table 3) A reduction of the phosphoramidite concentration
(and water concentration introduced by the amidite) slows degradation significantly
(entries 2, 3, 4). After 10 days at a phosphoramidite concentration of 0.05 M we
observe ca 3% degradation as compared to 8% at a concentration of 0.2 M. Additional
drying (aceotropically, vacuum) of the phosphoramidite had no effect on the solution
˚
stability (entries 5 and 6). However, the addition of molecular sieves (3A) to
phosphoramidite solutions provided significant benefit in terms of solution stability
(entry 7). The addition of triethylamine leads to a kinetic stabilization of the amidite
solution. As little as 5 mol-% with respect to the phosphoramidite reduces degradation
by more than 60% after a period of 10 days, providing similar protection in terms of
solution degradation as the addition of molecular sieves. To provide further evidence
for the proposed mechanism additional water was added to the phosphoramidite
solution (entries 11, 12, 13, 14). Interestingly, the extent of hydrolysis after 10 days is
only slightly increased in the sample to which 38 mol-% (with respect to 1c) water was
added. This supports the earlier notion that diisopropylamine formed during the

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773
Figure 3. (A) Formation of CE-H-phosphonate 2c of dG amidite 1c over time; (B) Formation of
H-phosphonate 3c of dG amidite 1c over time; (C) Formation of CE-phosphonoamidate 4c of dG
amidite 1c over time. (View this art in color at www.dekker.com.)
hydrolysis reaction protects unreacted amidite from further hydrolysis. However,
removal of water is the preferred mode of stabilization as any residual water reacts
rapidly with the phosphoramidite upon activation with 1H-tetrazole (1 acts as its own
drying agent!) prior to the coupling reaction of oligonucleotide synthesis. Depending on
the phosphoramidite excess used the coupling efficiency may be reduced, resulting in
lower yield.

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Krotz et al.
Table 3. Stability of dG amidite 1c in acetonitrile solution under various conditions (area-%
by HPLC).
HPLC analysis (day 10)
Entry
Experimental conditions
Control (day 0)
0.2 M
1c
2c
3c
4c
other
1
2
3
4
5
6
7
8
99.0
91.0
94.4
96.1
90.4
89.9
95.9
90.1
0.20
2.65
2.33
1.92
3.63
4.19
0.98
3.61
0.06
4.36
2.16
1.16
3.98
4.04
1.22
4.31
0.09
1.13
0.37
0.18
1.08
1.02
0.49
1.17
0.65
0.86
0.74
0.64
0.91
0.85
1.41c
0.81
0.1 M
0.05 M
Amidite vacuum dried (4 micron, 2 days)
Amidite dried aceotropically (benzene )a
b
˚
As entry 2, mol sieves 3A
As entry 2, triethylamine
(0.4 mg, 0.2 mol-%)
9
As entry 2, triethylamine
(2 mg, 1 mol-%)
92.1
96.3
2.67
0.62
3.48
1.69
0.98
0.68
0.77
0.71
10
As entry 2, triethylamine
(10 mg, 5 mol-%)
11
12
13
14
As entry 2, + water (3.5 mg, 10 mol-%)
As entry 2, + water (7 mg, 19 mol-%)
As entry 2, + water (10.5 mg, 29 mol-%)
As entry 2, + water (14 mg, 38 mol-%)
88.6
87.0
86.8
85.2
4.39
4.88
5.14
5.66
4.99
5.73
5.80
6.60
1.23
1.45
1.37
1.58
0.79
0.94
0.89
0.96
aA solution of 1c (3.0 g) in benzene (50 ml) was evaporated to dryness at 30°C, then kept under
vacuum (4 microns) for 2 days.
bMolsieves (1.0 g) was added to a solution of 1c (1.68 g, 2 mmol) in acetonitrile (10 ml).
cA slight increase of trialkyl phosphite triester species is observed.
CONCLUSION
In conclusion, we have shown that standard phosphoramidites 1a–d used in
oligonucleotide synthesis degrade in solution through a mechanism that involves acid-
catalyzed hydrolysis leading to CE-H-phosphonate 2, base-catalyzed elimination of
acrylonitrile of 2 leading to H-phosphonate 3 and an autocatalytic addition/elimination
Arbuzov-type reaction leading to CE-phosphonoamidate 4. The rate of degradation
decreases in the order dG > dA > dC ꢀ T. Consistent with the proposed degradation
mechanism amidite solutions may be stabilized by dilution, by addition of molecular
sieves or by addition of small amounts of base (e. g. triethylamine). Removal of
moisture is certainly the preferred method. Synthetic methods and isolation procedures
providing phosphoramidites with significantly lower (less than 0.01%) water content
are desirable.
REFERENCES
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4,725,677, February 6, 1988.

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2. Sinha, N.D.; Biernat, J.; Ko¨ster, H. b-Cyanoethyl N,N-dialkylamino/N-morpholi-
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Received September 29, 2003
Accepted March 22, 2004

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