New phosphorylating reagents for deoxyribonucleosides and oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2017.02.034

New phosphorylating reagents 1 and 2 were prepared in three steps from 4-hydroxybenzaldehyde. They showed good efficiency in the solid phase synthesis of 5′-phosphate monoester nucleosides. End-phosphate DNA sequence synthesis demonstrated the efficiency of the new reagents (1 and 2) according to the general procedure of automated DNA synthesis. The oxidation of P(III) to P(V) and the removal of benzyl protecting groups were achieved in a single step by treatment with a 0.02 M I₂/pyridine/H₂O solution. Due to this one-pot treatment, it is possible to use the phosphorylating reagents (1 and 2) for the synthesis of base-sensitive ODNs. The reagents 1 and 2 are unique among phosphorylating reagents.

Full text of DOI:10.1016/j.tetlet.2017.02.034

Accepted Manuscript
New phosphorylating reagents for deoxyribonucleosides and oligonucleotides
Valeria Romanucci, Armando Zarrelli, Annalisa Guaragna, Cinzia Di Marino,
Giovanni Di Fabio
PII:
S0040-4039(17)30195-8
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.02.034
TETL 48645
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Accepted Date:
16 January 2017
9 February 2017
Please cite this article as: Romanucci, V., Zarrelli, A., Guaragna, A., Di Marino, C., Di Fabio, G., New
phosphorylating reagents for deoxyribonucleosides and oligonucleotides, Tetrahedron Letters (2017), doi: http://
dx.doi.org/10.1016/j.tetlet.2017.02.034
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New phosphorylating reagents for
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deoxyribonucleosides and oligonucleotides
Valeria Romanucci, Armando Zarrelli,* Annalisa Guaragna, Cinzia Di Marino and Giovanni Di Fabio*

1
Tetrahedron Letters
New phosphorylating reagents for deoxyribonucleosides and oligonucleotides
Valeria Romanucci,a Armando Zarrelli,a,b,* Annalisa Guaragna,a Cinzia Di Marinoa,b and Giovanni Di
Fabioa
a Department of Chemical Sciences, University of Napoli 'Federico II', Via Cintia 4, 80126 Napoli, Italy
b Inter-University Consortium “SannioTech”, 82030 Apollosa, Benevento, Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
New phosphorylating reagents
1 and 2 were prepared in three steps from 4-
hydroxybenzaldehyde. They showed good efficiency in the solid phase synthesis of 5′-phosphate
monoester nucleosides. End-phosphate DNA sequence synthesis demonstrated the efficiency of
the new reagents (1 and 2) according to the general procedure of automated DNA synthesis. The
oxidation of P(III) to P(V) and the removal of benzyl protecting groups were achieved in a
single step by treatment with a 0.02 M I2/pyridine/H2O solution. Due to this one-pot treatment, it
is possible to use the phosphorylating reagents (1 and 2) for the synthesis of base-sensitive
ODNs. The reagents 1 and 2 are unique among phosphorylating reagents.
Available online
Keywords:
Phosphorylation
Phosphate protecting group
Nucleotides
2009 Elsevier Ltd. All rights reserved.
Oligonucleotides
Solid phase synthesis
Synthetic oligonucleotides (ONs) have found numerous
applications as tools and diagnostics in molecular biology. In this
context, 5'-phosphate ONs are requisite valuable tools for gene
construction, cloning, mutagenesis, the ligation chain reaction
and many other biological applications.1 Over the years, a
number of methods have been reported that allow chemical 3'-
/5’-phosphorylation for obtaining monoester derivatives of
nucleosides, ONs and other polyols.2 While the coupling
efficiency for some of these reagents cannot be easily monitored
because the reagent is devoid of marker groups, others require
elevated temperature conditions that generally are incompatible
oligonucleotides. The key point of our approach, which is based
on phosphoramidite chemistry, is the design of new
a
phosphorous protecting group that is removable during the
oxidation step of P(III) to P(V) by treatment with a conventional
oxidizer solution (I2/pyridine/H2O). The phosphorylating agents 1
and 2 (Scheme 1) were designed to satisfy the above
requirements and were prepared similarly to 3, which was the
subject of a patent5 based on a new phosphorylating reagent for
the synthesis of ODN 5'-phosphate.
The phosphorylating agents 1 and 2 were prepared in three
steps from 4-hydroxybenzaldehyde (Scheme 1) in 55% overall
yield.6,7
with
the
synthesis
of
modified
ONs
and
oligodeoxyribonucleotides (ODNs). In fact, this aspect is crucial
for the preparation of ODNs functionalized with thermo- and
base-sensitive groups (e.g., phosphotriester groups).3 In 2005,
Ausìn C. et al.4 reported a new phosphorylating reagent useful for
generating phosphate monoesters in the presence of
phosphorothioate triester functions with minimal cleavage of the
functions (<2%). In this work, the phosphorus protecting group
was mercaptoethanol, and its removal required very peculiar
basic conditions (ca 5 bar MeNH2 gas for 30 minutes), which are
borderline with the stability of ester and phosphotriester
functions. The synthesis of a phosphorylating agent with
removable protecting groups in very mild conditions, to be
compatible with thermo- and base-sensitive functions, is still in
great demand.
In this work, we have focused our attention on the
development of new phosphorylating reagents that are totally
Scheme 1. a. MMTrCl (or TrCl for 7), DIEA in DCM, 6 h; b. NaBH4 in
THF, 6 h; c. i-Pr2NPCl2, DIEA in DCM, 1.5 h.
compatible with the preparation of functionalized base-sensitive
———
 Corresponding authors. Tel.: +39-081-674001; e-mail: difabio@unina.it; Tel.: +39-081-674472; e-mail: zarrelli@unina.it

2
Tetrahedron
The commercially available 4-hydroxybenzaldehyde 4 was
4-MMTr-benzyl group. We have explained this behaviour with a
one-pot mechanism of oxidation/deprotection similar to that
reported by Filippov, D. V. et al.9 The same results were also
observed for the reagent 3.
first reacted with 4-monomethoxytritylchloride (MMTrCl) or
tritylchloride (TrCl) in dry DCM and in the presence of DIPEA,
affording 5 and 7 in 82% and 75% yields, respectively. After
reduction with NaBH4 in THF, alcohols 6 and 8 were obtained in
80% and 85% yields, respectively. 7 (or 8) was then reacted with
i-Pr2NPCl2 and DIPEA in anhydrous DCM, and after silica gel
chromatography, 1 (or 2) was obtained in a good yield (82% for
16 and 80% for 27). The identities of all desired products were
confirmed by NMR and ESI-MS analyses.
To further assess the goodness of 1 and 2 as end-
phosphorylating reagents, the preparation of an ON 5′-phosphate
model was undertaken (Scheme 2). Specifically, the solid-phase
syntheses of Pd(CAGT) and d(CAGTCAGTCAGTCAGT) were
P
performed according to an automated synthesis protocol. ODNs
were released from the support by NH4OH treatment, and RP-
HPLC analyses indicate that the use of 1 or 2 led to the formation
of ODN 5′-phosphate in yields exceeding 90%. MALDI-TOF
analysis confirmed the expected structures of 11 and 12.
The conditions for the optimal coupling efficiency of 1 (or 2)
were first investigated by performing manual synthesis of 5′-
phosphate monoester derivatives. For this purpose, CPG
anchored the 3’-O-succinyl (9a-d, Scheme 2) contained in a
classic tube for the synthesis of DNA, which was mixed with
0.25 M 1H-tetrazole in MeCN (activator solution) for 2 minute
and then with a solution of 0.1 M phosphoramidite 1 (or 2) in
MeCN for 2 minutes. This process was repeated three times,
performing all operations with the same solvents, instrumentation
and water-free conditions.
The removal of phosphate protecting groups in very mild
conditions was then evaluated in an ad hoc experiment (Scheme
3) by the use of nucleotide model 13 (3'-O-benzoyl-N6-benzoyl-
2'-deoxyadenosine). Condensation of 1 with 13 in the presence of
1H-tetrazole in MeCN, followed by silica gel chromatography,
afforded the nucleoside phosphite triester 14 (Scheme 3) in 75%
yield.10
Scheme 3. Synthesis of dA 5’-monophospate derivative 15: oxidation and
deprotection of phosphorus in a one-pot reaction with I2/pyridine/H2O.
Scheme 2. Synthesis of 5’-phosphate nucleoside monoesters and end-
phosphate ODNs.
Treatment of 14 with 0.02 M I2/pyridine/H2O in THF was
performed. The reaction was monitored by 31P-NMR, and after
only a few minutes (<5 minutes), the disappearance of the typical
signal of the phosphite at 140 ppm and the appearance of its
monoester phosphate at –0.7 ppm were observed. The crude
material was then purified by silica gel chromatography, and 2’-
deoxy adenosine-5’-monophosphate nucleoside 15 (Scheme 3)
was isolated in 60% yield. As expected, 31P-NMR analysis
showed the diagnostic variation of the phosphorus chemical shift
Subsequently, the resins were washed with MeCN and then
treated with a 1.1 M solution of tert-butyl-hydroperoxide
(tertButOOH) in decane for 4 minutes. After the oxidation
treatment, the resins were washed with MeCN and then
submitted to a spectrophotometric test of the trityl cation for
calculation of the reaction yield. The resultant yields were on
average 95%. The treatment of the resins with 2% DCA in DCM
allowed the removal of the MMTr group. Finally, treatment with
a solution of 28% aqueous ammonia at 50 °C for 5 hours allowed
the deprotection and detachment of the desired nucleotides from
the supports. RP-HPLC analyses of the crude detached materials
were performed. The HPLC profiles of 10a-d were identical to
those of 5′-monophosphate nucleosides obtained from
commercial sources.8
1
from 140 to –0.7 ppm, and H,13C NMR and ESI-MS analyses
confirmed the expected structure of 15.
Conclusion
New phosphorylating reagents 1 and 2 were prepared in good
yields starting from 4-hydroxybenzaldehyde (Scheme 1), and
their efficiency in the synthesis of 5′-phosphate monoester
nucleosides (10a-d, Scheme 2) has been demonstrated.
Phosphorylating reagents 1 and 2 are also efficient in the
preparation of end-phosphate ODNs (11 and 12 Scheme 2). Thus,
following the classic procedure of automated DNA synthesis, the
end-phosphate DNA sequence models were synthesized in very
good yields. The removal of phosphate protective groups in very
mild conditions by an ad hoc experiment on nucleoside model 13
Surprisingly, when the oxidation step was performed with the
classic oxidizer solution (0.02 M I2/pyridine/H2O) for 10 minutes,
the MMTr cation tests on aliquots of weighted resin provided
values close to zero. The treatment of the resulting support with a
solution of 28% aqueous ammonia at 50 °C for 5 hours led to the
5'-end phosphorylated nucleosides (10a-d), as shown by HPLC
analysis.8 In this case, the oxidation treatment led not only to the
oxidation of phosphite to phosphate but also the removal of the

3
desired product (1, 87%). 1H NMR (400 MHz, 25 °C, , ppm in
was demonstrated (Scheme 3). The oxidation to phosphate and
the removal of phosphate protecting groups were achieved by a
one-pot treatment with a 0.02 M I2/pyridine/H2O solution
(oxidizer solution). In light of these results, the new
phosphorylating agents 1 and 2 are compatible with base-
sensitive functions and would be very useful for the synthesis of
phosphate monoesters endowed with base-labile functions
(esters, phosphotriester). This makes the reagents 1 and 2 unique
among all phosphorylating reagents.
CDCl3): δ 7.60 (d, J = 7.6 Hz, 8H), 7.42 (d, J = 8.8 Hz, 4H), 7.15-
6.98 (complex signals, 16H), 6.82 (d, J = 7.6 Hz, 4H), 6.61 (d, J =
8.8 Hz, 4H), 4.48 (m, 4H), 3.55 (m, 2H), 3.20 (s, 6H), 1.06 (d, J =
6.8 Hz, 12H). 13C NMR (100 MHz, , 25 °C, , ppm in CDCl3): δ
158.8, 155.9, 145.1, 135.7, 132.8, 130.8, 127.9, 127.7, 127.4,
126.8, 120.9, 113.0, 90.5, 65.1, 54.3, 42.9, 24.3. 31P NMR (162
MHz , 25 °C, , ppm in CDCl3): d 147.8. ESI-MS calcd. [M]
921.09; found 945.30 [MNa]+.
Synthesis of 2 (Scheme 1): Starting from 4, the same procedure
was followed using the same molar ratios and the same
purification conditions used to synthesize 1. The phosphoramidite
2 was obtained in an 80% yield. 1H NMR (400 MHz, 25 °C, ,
ppm in CDCl3): δ 7.53 (d, J = 7.1 Hz, 12H), 7.35-7.23 (complex
signals, 18H), 7.01 (d, J = 8.4 Hz, 4H), 6.70 (d, J = 8.5 Hz, 4H),
4.54 (m, 4H), 3.64 (m, 4H), 1.17 (d, J = 6.8 Hz, 12H). 13C NMR
(100 MHz, 25 °C, , ppm in CDCl3): δ 155.6, 144.2, 132.2, 132.2,
129.0, 127.8, 127.5, 127.2, 90.3, 65.2, 65.0, 43.0, 42.9, 24.7, 24.6
31P NMR (162 MHz , 25 °C, , ppm in CDCl3):  147.3. ESI-MS
calcd. [M] 861.39; found 885.20 [MNa]+.
7.
Acknowledgments
We acknowledge AIPRAS Onlus (Associazione Italiana per la
Promozione delle Ricerche sull’Ambiente e la Saluta umana) for
financial support. We also thank Tecno Bios® for the HPLC
facilities.
8.
9.
See Supporting Information file.
References and notes
van der Heden van Noort, G. J.; Verhagen, C. P.; van der Horst,
M. G.; Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V.
Org. Lett. 2008, 10, 4461–4464.
1.
2.
Khakshoor, O.; Kool, E. T. Chem. Commun 2011, 47, 7018-7024,
and references therein.
Szczepanik, M. B.; Désaubry, L.; Johnson, R. A. Tetrahedron
Lett. 1998, 39, 7455-7458; Horn, T.; Urdea, M. S. Tetrahedron
Lett. 1986, 27, 4705-4708; Olesiak, M.; Krajewska, D.;
10. Synthesis of 15 (Scheme 3): 35 mg (0.076 mmol) of 3'-O-benzoyl-
N6-benzoyl-2'-deoxyadenosine 13 was dissolved in 300 L of
DCM, after which 500 L of activator solution was added (1H-
tetrazole 0.25 M), and finally a solution of 76 mg of 1 (0.083
mmol) dissolved in 500 L of DCM was added. After 30 min, the
reaction was completed as shown from TLC [hexane/EtOAc 40:60
(v/v)] control. The solvent was removed under vacuum, and the
crude material was purified by column chromatography on silica
gel in hexane/EtOAc 50:50 (v/v) with 2% of TEA. From the
column was recovered 73 mg of clean desired product 14 (75%).
1H NMR (400 MHz, 25 °C, , ppm in CDCl3): δ 8.84 (s, 1H), 8.39
(s, 1H), 8.10 (d, J =8.4 Hz, 2H), 8.05 (d, J = 8.5 Hz, 2H), 7.66-
7.17 (complex signals, 30H), 6.94 (m, 4H), 6.76 (d, J = 9.0Hz,
4H), 6.73 (d, J = 9Hz, 4H), 5.57 (d, J = 5.6Hz, 1H), 4.40 (m, 1H),
4.03 (complex signals, 2H), 3.75 (m, 1H), 2.63 (m, 2H). 13C NMR
(100 MHz, , 25 °C, , ppm in CDCl3): δ 165.8, 158.5, 156.2,
152.8, 151.7, 149.5, 144.5, 141.2, 135.7, 133.6, 132.8, 129.8,
128.9, 128.7, 128.2, 128.1, 127.7, 127.0, 120.8, 112.9, 90.2, 84.6,
84.2, 76.1, 64.7, 64.5, 64.4, 64.3, 60.4, 55.1. 31P NMR (162 MHz,
25 °C, , ppm in CDCl3):  140.4. ESI-MS calcd. [M] for
C78H66N5O11P = 1279.45; found 1303.69 [MNa]+.
Wasilewska, E.; Korczyn´ski, D.; Baraniak, J.; Okruszek, A.; Stec,
W. J. Synlett 2002, 967-971; Tosquellas, G.; Alvarez, K.;
Dell’Aquila, C.; Morvan, F.; Vasseur, J.-J.; Imbach, J.-L.; Rayner,
B. Nucleic Acids Res. 1998, 26, 2069-2074; Guzaev, A.; Salo, H.;
Azhayev, A.; Lönnberg, H. Tetrahedron 1995, 51, 9375-9384;
Lartia, R.; Asseline, U. Tetrahedron Lett. 2004, 45, 5949-5952.
Grajkowski, A.; Pedras-Vasconcelos, J.; Wang, V.; Ausín, C.;
Hess, S.; Verthelyi, D.; Beaucage, S. L. Nucleic Acids Res. 2005,
33, 3550–3560 and references therein.
3.
4.
Ausìn, C.; Grajkowski, A.; Cieślak, J.; Beaucage, S. L. Org. Lett.
2005, 7, 4201-4204.
5.
6.
Di Fabio, G; Zarrelli, A. Patent IT 0001420344, 2015.
Synthesis of 1 (Scheme 1): The 4-hydroxybenzaldehyde 4 (0.8 g,
6.5 mmol) was reacted with 4-monometoxytriphenylmethyl (1.0 g,
3.2 mmol) in 10 mL of anhydrous DCM in the presence of DIEA
(2.2 mL, 12.9 mmol). After 6 hours at r.t., the reaction was
quenched by dilution with DCM (100 mL), and the organic phase
was washed three times with a solution of 0.1 M NaOH (3x100
mL). The organic phase was dried with MgSO4, and then the
solvent was removed under vacuum. The crude material was then
purified on a column of silica gel (70 g) suspended in
3 mL of oxidizer solution (0.02 M I2/pyridine/H2O) was added to
70 mg (0.05 mmol) of 14 and stirred. The reaction was followed
by 31P NMR by suspending a small amount of crude material in
CDCl3. Five minutes after the addition of the oxidizer solution, the
disappearance of the signal at 140 ppm (phosphite triester) and the
appearance of a typical signal of a phosphate monoester at -0.7
ppm were observed. The solvent was removed under vacuum, and
the crude material was purified by silica gel chromatography in
DCM/MeOH 80:20 (v/v). From the column was recovered 19 mg
of 3'-O-benzoyl-N6-benzoyl-2’-deoxyadenosine-5'-
hexane/EtOAc 70:30 (v/v) with 1% of TEA, leading to product 5
(1.05 g, 82%). 1H NMR (400 MHz, 25 °C, , ppm in CDCl3): δ
9.76 (s, 1H), 7.57-7.25 (complex signals, 14H), 6.86 (d, J = 8.5
Hz, 2H), 6.82 (d, J = 8.0 Hz, 2H), 3.77 (s, 3H). 13C NMR (100
MHz, 25 °C, , ppm in CDCl3): δ 190.7, 161.9, 158.7, 143.8,
134.9130.6, 130.2, 128.3, 127.8, 127.3, 120.3, 113.1, 91.1, 55.0.
Product 5 (1.05 g, 2.66 mmol) was subsequently treated with 0.25
g of NaBH4 (6.60 mmol) in THF (10 mL) for 6 hours at r.t. The
mixture was diluted with DCM (3x100 mL), and the organic phase
was washed three times with water (100 mL). The organic phase
was dried with MgSO4, and then the solvent was removed under
vacuum. The crude solid thus obtained was purified on a column
of silica gel (70 g) suspended in hexane/EtOAc 60:40 (v/v) with
1% of TEA. From the column was recovered 0.845 g of clean
desired product 6 (2.13 mmol, 80%). 1H NMR (400 MHz, 25 °C,
, ppm in CDCl3): δ 7.49 (d, J=6.0 Hz, 4H), 7.37-7.22 (complex
signals, 8H), 6.98 (d, J = 7.2 Hz, 2H), 6.79 (d, J = 7.2 Hz, 2H),
6.70 (d, J = 6.8 Hz, 2H), 4.45 (s, 2H), 3.75 (s, 3H). 13C NMR (100
MHz, 25 °C, , ppm in CDCl3): δ 158.4, 155.8, 144.5, 135.6,
133.3, 130.4, 128.6, 127.6, 127.4, 126.9, 120.6, 112.8, 90.0, 64.8,
55.0. Then, 0.40 g (1.01 mmol) of product 6 was reacted with
N,N-diisopropyldichlorophosphoramidite (124 L, 0.67 mmol) in
the presence of DIEA (348 L, 2.68 mmol) in DCM (7 mL). After
1.5 h, the reaction was quenched by dilution with DCM, and the
organic phase was washed three times with cold water. The
organic phase was dried with MgSO4 and the solvent removed
under vacuum. The material was purified with column
monophosphate desired product 15 (60%). 1H NMR (400 MHz, 25
°C, , ppm in CDCl3): δ 8.89 (s, 1H), 8.78 (s, 1H), 8.13-7.36
(complex signals, 4H), 7.77-7.43 (complex signals, 4H), 6.69 (dd,
J = 8.4 and 6.1 Hz, 1H), 5.69 (m, 1H), 4.46 (m, 1H), 4.02 (m, 2H),
3.01 (m, 1H), 2.79 (dd, J = 14.0, 4.0 Hz, 1H). 13C NMR (100
MHz, , 25 °C, , ppm in CDCl3): δ 167.6, 154.1, 153.6, 151.4,
144.5, 135.1, 134.4, 131.0, 130.1, 130.0, 129.8, 124.1, 86.2, 84.0,
77.6, 66.8, 39.2. 31P NMR (162 MHz , 25 °C, , ppm in CDCl3): 
– 0.7. ESI-MS calcd. [M] for C24H22N5O8P = 539.12; found
562.66 [MNa]+; 578.83 [MK]+.
chromatography of silica gel in hexane/EtOAc 85:15 (v / v) with
2% of TEA. From the column was recovered 0.74 g of clean

4
Tetrahedron
1. New phosphorylating reagents
2. New phosphate protecting group
3. Synthesis of Nucleotides and Oligonucleotides by
Solid phase synthesis