The synthesis of amino acid methyl ester 5′-phosphoamidates of protected uridine

Article abstract of DOI:10.1080/10426500903490237

In this article, we used the Atherton-Todd reaction to synthesize amino acid methyl ester 5′-phosphoamidates of uridine as prodrugs. Their structures were confirmed by 1H NMR, 31P NMR, 13C NMR, IR, and mass spectrometry. Supplemental materials are availab

Full text of DOI:10.1080/10426500903490237

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The Synthesis of Amino Acid Methyl
Ester 5′-Phosphoamidates of Protected
Uridine
W. Z. Chen a b & Y. F. Zhao a c
a Department of Chemistry and Key Laboratory for Chemical Biology
of Fujian Province , Xiamen University , Xiamen, China
b The Third Institute of Oceanography of the State Oceanic
Administration , Xiamen, China
c Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical
Biology (Ministry of Education), Department of Chemistry , Tsinghua
University , Beijing, China
Published online: 24 Sep 2010.
To cite this article: W. Z. Chen & Y. F. Zhao (2010) The Synthesis of Amino Acid Methyl Ester 5′-
Phosphoamidates of Protected Uridine, Phosphorus, Sulfur, and Silicon and the Related Elements,
185:10, 2054-2063, DOI: 10.1080/10426500903490237
To link to this article: http://dx.doi.org/10.1080/10426500903490237
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Phosphorus, Sulfur, and Silicon, 185:2054–2063, 2010
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500903490237
THE SYNTHESIS OF AMINO ACID METHYL ESTER
5ꢁ-PHOSPHOAMIDATES OF PROTECTED URIDINE
W. Z. Chen1,2 and Y. F. Zhao1,3
1Department of Chemistry and Key Laboratory for Chemical Biology of Fujian
Province, Xiamen University, Xiamen, China
2The Third Institute of Oceanography of the State Oceanic Administration,
Xiamen, China
3Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology
(Ministry of Education), Department of Chemistry, Tsinghua University, Beijing,
China
In this article, we used the Atherton–Todd reaction to synthesize amino acid methyl ester
1
5ꢁ-phosphoamidates of uridine as prodrugs. Their structures were confirmed by H NMR,
31P NMR, 13C NMR, IR, and mass spectrometry.
Supplemental materials are available for this article. Go to the publisherꢁs online edition of
Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental
file.
Keywords Amino acid methyl ester 5ꢀ-phosphoamidates of protected uridine; Atherton–Todd;
spectra characterization; synthesis
INTRODUCTION
Nucleosides and their analogues as potential anticancer and antivirus agents have
been studied extensively in recent years.1–3 For example, several purine and pyridine bases
and nucleoside analogs were used as a chemotherapeutic arsenal. However, the biological
activity of most analogs requires intracellular metabolism to 5ꢀ-mononucleotides by kinase.4
In order to overcome the problem of drug resistance and improve membrane penetration, a
series of amino acid phosphoramidate di- and tri-esters of nucleotides have been developed
to deliver phosphorylated nucleoside analogues as neutral derivatives into the cell.5 These
types of compounds exhibit enhancing antiviral activity and reducing cytotoxicity as com-
pared to the corresponding parent nucleosides.6,7 For the purpose of developing new types
of prodrugs, a new type of amino acid methyl ester 5ꢀ-phosphoamidates of nucleoside was
synthesized, and is reported in this article.
Received 17 September 2009; accepted 13 November 2009.
Address correspondence to Y. F. Zhao, Department of Chemistry and Key Laboratory for Chemical Biology
of Fujian Province, Xiamen University, Xiamen 361005, China. E-mail: yfzhao@xmu.edu.cn
2054

AMINO ACID METHYL ESTER 5ꢁ-PHOSPHOAMIDATES
2055
RESULTS AND DISCUSSION
Synthesis
The reported synthesis of O-nucleoside N-phosphoryl amino acid methyl esters
involved the coupling of amino acid methyl esters with nucleoside monophosphates in
the presence of condensing reagents such as dicyclohexylcarbodiimide (DCC) and 2,4,6-
triisopropylbenzenesulfonyl chloride,8 or with nucleoside phosphite triesters in the presence
of iodine.9 Phosphorochloridate approach was also reported in the preparation of aryl phos-
phate derivative of nucleoside analogs.10 The amino acids chosen, however, were usually
the nonpolar ones without active side chains. In the present article, we report the application
of the Atherton–Todd11 reaction in the synthesis of O-alkyl O-2ꢀ,3ꢀ-isopropylideneuridine
N-phosphoryl amino acid methyl ester (Scheme 1). The present methodology was applica-
ble to amino acid methyl esters with different side chains and with either of the nucleosid-3ꢀ
(or 5ꢀ)-yl H-phosphonates. Owing to the low nucleophilic reactivity of amino groups on the
nucleobases and the hydroxyl on both the amino acid and the nucleoside compared with
the α-amino group, it was unnecessary to protect them in the coupling reaction.
O
N
O
N
NH
NH
O
O
Cl
P O
Cl
O
O
ii
iii
i
Cl P Cl
Cl
+
HO
O
O
O
O
O
N
O
1
2
NH
NH
O
N
O
O R
O
O
iv
O
O
H
H3C O C C N P O
HP O
O
H
O
O
O
O
O
3
4 a R=H
b R=CH3
c R=CH(CH3)2
d R=CH2CH(CH3)2
e R=CH2OH
f
R=CH2COOH
g R=CH2Ph
h R=CH2PhOH
NH
i
R=H2C
N
j
R=CH2CH2SCH3
Scheme 1 Synthetic route of amino acid methyl ester 5ꢀ-phosphoamidates of protected uridine. Reagents and
conditions: (i) NEt3, dry CH2Cl2, 0◦C for 0.5 h, then room temperature for 3 h; (ii) dry CH2Cl2, menthol, tert-butyl
alcohol, 0◦C, then room temperature for 0.5 h; (iii) NEt3, dry CH2Cl2, 0◦C, then room temperature for 10 min;
(iv) CCl4, NEt3, H2O, CH3CN, amino acid methyl ester, room temperature.

2056
W. Z. CHEN AND Y. F. ZHAO
31P NMR Spectra
The 31P nucleus is a very sensitive probe for structural studies of organophos-
phorus compounds. The 31P NMR spectra of 2ꢀ,3ꢀ-isopropylidenenucleoside-5ꢀ-O-yl-H-
phosphonate and O-alkyl O-2ꢀ,3ꢀ-isopropylideneuridine N-phosphoryl amino acid methyl
ester are in the range of 5 to about 10 ppm. Due to the chirality at the phosphorus center,
they both exhibit two different chemical shift values in the 31P NMR spectra (Figures 1a
and 1b), and the integral areas of 31P NMR are basically the same if protons are decoupled.
But if the protons are not decoupled, nucleoside-5ꢀ-O-yl-H-phosphonate (Figure 1a)
has a distinct different chemical shift value from uridine N-phosphoryl amino acid methyl
ester (Figure 2a). It exhibits four different chemical shift values in the 31P NMR spec-
tra. This is because the phosphorus in nucleoside-5ꢀ-O-yl-H-phosphonate was coupled
1
by the hydrogen on the phosphorus. The coupling constant JPH is about 700 Hz in
1
nucleoside-5ꢀ-O-yl-H-phosphonate. For example, the coupling constant JPH is 703 Hz
in menthyl 2ꢀ,3ꢀ-isopropylideneuridin-5ꢀ-yl-H-phosphonate (3). But in O-alkyl O-2ꢀ,3ꢀ-
isopropylideneuridine N-phosphoryl amino acid methyl ester, although the hydrogen on
2
1
the nitrogen has a coupling effect on the phosphorus, JPH is much smaller than JPH
,
and the coupling splitting is not distinct. This is the difference between 31P NMR spec-
tra of nucleoside-5ꢀ-O-yl-H-phosphonate and O-alkyl O-2ꢀ,3ꢀ-isopropylideneuridine N-
phosphoryl amino acid methyl ester.
1H NMR Spectra
2ꢀ,3ꢀ-Isopropylideneuridine-5ꢀ-O-yl-H-phosphonate and O-alkyl O-2ꢀ,3ꢀ-isopropyli
deneuridine N-phosphoryl amino acids methyl esters are a pair of diastereoisomers due
1
to the chirality at the phosphorus center. Their H NMR spectra are all pairs of peaks,
Figure 1 The 31P NMR spectrum of menthyl 2ꢀ,3ꢀ-isopropylideneuridin-5ꢀ-yl-H-phosphonate (3).

AMINO ACID METHYL ESTER 5ꢁ-PHOSPHOAMIDATES
2057
Figure 2 The 31P NMR spectrum of O-nucleoside N-phosphoryl amino acids methyl esters (4a).
especially the H-6 of the nucleoside and methyl ester of the amino acid. For example,
OCH3 on the compound 4h exhibits two single peaks. Their chemical shift values are 3.71
and 3.70.
13C NMR Spectra
Due to the chirality at the phosphorus center, 2ꢀ,3ꢀ-isopropylideneuridine-5ꢀ-O-yl-H-
phosphonate and O-alkyl O-2ꢀ,3ꢀ-isopropylideneuridine N-phosphoryl amino acids methyl
esters are a pair of diastereoisomer, so some carbon atoms exhibit double peaks in the
13C NMR spectra. For example, C-6 on the compound 4h exhibits two single peaks. Their
chemical shift values are 141.69 and 142.00.
Mass Spectra
Positive-ion mode electrospray ionization (ESI) mass spectral fragmentation path-
ways of 2ꢀ,3ꢀ-isopropylideneuridine phosphates were analyzed by multistage tandem mass
spectrometry. Tables S1 and S2 (Supplemental Materials, available online) list the fragment
ions observed in multistage tandem mass spectra of the protonated and sodium ion adducts
of 4a–j, respectively.
Because these compounds displayed very similar fragmentation patterns in positive-
ion mode ESI multistage tandem mass spectrometry, the mass spectral fragmentation path-
ways of compound 4h were discussed as a typical example (Scheme S1, Supplemental
Materials). The protonated molecule has richer fragmentation pathways than the sodium

2058
W. Z. CHEN AND Y. F. ZHAO
ion adduct. The abundant characteristic fragment ions were observed by loss of uracil
(112), amino acid methyl ester (195), menthene (138), and acetone (58), corresponding to
the cleavage of glycosyl C-N between uracil and sugar, P-N, and C-O bonds. The fragment
ions almost always contained a phosphoryl group. In addition, it was interesting that the
fragmentation pathways of the sodium ion adduct showed some differences from that of
the protonated molecule. The methoxy group of amino acid methyl ester could migrate
from the carbonyl group to the phosphoryl group only for the sodium ion adduct, but not
for the protonated molecule [M + H]+. The ion at m/z 252 in the sodium ion adduct of
4h might yield in this way.12 The reason is that metal ions such as the sodium ion could
coordinate with both carbonyl oxygen and phosphoryl oxygen to form a seven-membered
ring, which might help the attack of the methoxy group on phosphorus. In contrast, with
the smaller size, a proton could only attach to either the phosphoryl oxygen or the carbonyl
oxygen. These characteristic ions in ESI mass spectra and the rearrangement mechanism
are useful for the structural determination of amino acid methyl ester 5ꢀ-phosphoramidate
of nucleosides.
CONCLUSION
In summary, we use a convenient and efficient approach to synthesize a series of
amino acid methyl ester 5ꢀ-phosphoamidates of protected uridine under mild conditions.
The reaction process was traced by 31P NMR. In addition, the structures of these compounds
were verified and fragmentation pathways were analyzed by positive-ion mode ESI-MSn.
EXPERIMENTAL
Material and Methods
All glassware was dried in an oven for at least 3 h at 140 ◦C prior to use. Air-sensitive
materials were transferred under a nitrogen atmosphere. CH2Cl2 and NEt3 were dried over
1
CaCl2 and KOH, respectively. PCl3 was distilled. H NMR and 13C NMR spectra were
recorded in a Bruker AM 500 spectrometer. TMS (δ = 0.0) and CDCl3 (δ = 7.24 ppm)
1
were references for H and 13C NMR spectra, respectively. 13C NMR spectra were all
taken under 1H-decoupled and 31P-coupled conditions. 31P NMR spectra were taken on a
Bruker AM 500 spectrometer under 1H decoupled conditions. 31P NMR chemical shifts are
reported in ppm downfield (+) or upfield (−) from external 85% H3PO4 as reference. ESI
were determined with a Bruker ESQYIRE 3000 plus. Operating conditions for ESI in the
positive ion mode were as follows: spray voltage, 4000 V; capillary temperature, 300◦C;
dry gas (N2), 10 L/min; nebulizer (N2), 30 psi. HRMS was recorded on a Micromass-LCT
Premier Time of Flight (TOF) mass spectrometer (Waters, USA).
Synthesis of Amino Acid Methyl Ester Hydrochloride
and Protected Nucleoside (1)
The preparations of amino acid methyl ester hydrochloride and protected nucleoside
were carried out according to the literature.13,14 All physical constants and spectroscopic
data of the products synthesized were in agreement with the literature.

AMINO ACID METHYL ESTER 5ꢁ-PHOSPHOAMIDATES
2059
General Procedure for Synthesis of Aryl Phosphoramidate
Derivatives of Nucleosides15
To a solution of PCl3 (10 mmol) in anhydrous dichloromethane (10 mL), 2ꢀ,3ꢀ-
isopropylideneuridine (1) was added (1 mmol) at −30◦C under a nitrogen atmosphere, and
the solution was stirred for 30 min at this temperature and then for 3 h at room tempera-
ture. The solvent and excess of PCl3 were removed under reduced pressure. The residue
was dissolved in anhydrous dichloromethane (10 mL). Menthol and tert-butyl alcohol
(2.5 mmol, 1:1) in anhydrous dichloromethane (5 mL) was then added dropwise to the
solution at 0◦C, and the reaction was completed within 30 min. A solution of triethylamine
(2 mmol) in anhydrous dichloromethane (5 mL) was added to the resulting solution. After
10 min, the solvent was removed by rotary evaporation, and the product was purified by
chromatography on silica gel using CH2Cl2–MeOH (50:1) as eluent. The final product
2ꢀ,3ꢀ-isopropylideneuridine-5ꢀ-O-yl-H-phosphonate (3) was obtained in 90% yield.
To a solution of amino acid ester (1.2 mmol), water (0.5 mL), tetrachloromethane
(0.5 mL), triethylamine (0.5 mL), and acetonitrile (5 mL), a solution of 2ꢀ,3ꢀ-
Isopropylideneuridine-5ꢀ-O-yl-H-phosphonate (3) (1 mmol) in acetonitrile (5 mL) was
added dropwise with an ice-bath. The solution was then stirred at room temperature for 10
min. The solvent was removed by rotary evaporation to a gum below 40 ◦C. The residual
syrup was subjected to silica gel column chromatography by elution with 5% methanol
in chloroform. Pooling and evaporation of appropriate fractions gave amino acid methyl
ester 5ꢀ-phosphoamidates of protected uridine 4a–j in 81–94% yields.
Menthyl 2ꢁ,3ꢁ-osopropylideneuridin-5ꢁ-yl H-phosphonate (3). Yield 4.37 g
(90%). IR (film) v: 3193, 3058, 2431, 1695, 1624, 1249, 1063, 954 cm−1 31P NMR (CDCl3,
.
202 MHz): δ 8.89, 8.04. 1H NMR (CDCl3, 500 MHz): δ 9.40 (br, 1H, H-3), 7.52, 6.12 (d,
d, 1H, JPH = 703.0 Hz, P-H), 7.27, 7.26 (d, d, 1H, J = 8.0 Hz, H-6), 5.65–5.67 (m, 2H,
H-1ꢀ, H-5), 4.86–4.87 (m, 1H, H-2ꢀ), 4.75–4.82 (m, 1H, H-3ꢀ), 4.19–4.26 (m, 4H, H-4ꢀ,
H-5ꢀ, OCHR), 2.06–2.13 (1H, m, Hmenthyl), 1.92–2.01 (1H, m, Hmenthyl), 1.81–1.90 (1H, m,
Hmenthyl), 1.58–1.60 (2H, m, Hmenthyl), 1.49 (s, 3H, >CCH3), 1.36–1.40 (1H, m, Hmenthyl),
1.27 (s, 3H, >CCH3), 1.12–1.19 (2H, m, Hmenthyl), 0.87–1.00 (1H, m, Hmenthyl), 0.84 (d, 6H,
J = 7.0 Hz, 2CH3), 0.72 (d, 3H, J = 7.0 Hz, CH3). 13C NMR (CDCl3, 100 MHz): δ 163.63
(C-4), 150.18 (C-2), 142.12 (C-6), 114.70 (>CMe2), 102.79, 102.69 (C-5), 94.27, 94.17
(C-1ꢀ), 85.63, 85.53, 85.47 (C-4ꢀ), 84.35 (C-2ꢀ), 80.73, 80.65 (C-3ꢀ), 78.95, 78.89 (OCHR),
64.82 (C-5ꢀ), 48.24 (Cmenthyl), 43.28, 42.79 (Cmenthyl), 33.82 (Cmenthyl), 31.52(Cmenthyl), 27.08
(Cmenthyl), 25.87, 25.69, 25.22 (CH3), 22.86 (Cmenthyl), 21.87, 21.85 (CH3), 20.84, 20.81
(CH3), 15.65 (CH3); HRMS (ESI) calcd for C22H36N2O8P: 567.2621, found 567.2616.
Glycine ester menthyl phosphoramidates of 2ꢁ,3ꢁ-isopropylideneuridine
(4a). Yield 496 mg (86.6%). IR (film) v: 3231, 1751, 1696, 1618, 1217, 1068, 990 cm−1
.
31P NMR (CDCl3, 202 MHz): δ 8.12, 7.32. 1H NMR (CDCl3, 500 MHz): δ 9.46, 9.40 (br,
br, 1H, H-3), 7.38, 7.34 (d, d, 1H, J = 8.0 Hz, H-6), 5.72–5.76 (m, 1H, H-1ꢀ), 5.66 (d, J =
8.0 Hz, 1H, H-5), 4.79–4.86 (m, 2H, H-2ꢀ, H-3ꢀ), 4.24–4.32 (m, 1H, OCHR), 4.06–4.23 (m,
3H, H-4ꢀ, H-5ꢀ), 3.68, 3.67 (s, s, 3H, OCH3), 3.39–3.65 (m, 3H, P-NH, H-α,), 2.13–2.20
(m, 1H, Hmenthyl), 1.92–1.98 (m, 2H, Hmenthyl), 1.58–1.60 (m, 2H, Hmenthyl), 1.51 (s, 3H,
>CCH3), 1.33–1.44 (m, 1H, Hmenthyl), 1.28 (s, 3H, >CCH3), 1.16–1.25 (m, 2H, Hmenthyl),
0.89–1.01 (m, 1H, Hmenthyl), 0.84 (d, 6H, J = 6.5 Hz, 2CH3), 0.74 (t, 3H, J = 7.0 Hz, CH3).
13C NMR (CDCl3, 125 MHz): δ 171.16 (COOMe), 163.59 (C-4), 150.11 (C-2), 141.69,
141.33 (C-6), 114.09 (>CMe2), 102.27 (C-5), 92.64, 93.15 (C-1ꢀ), 85.45, 84.66 (C-4ꢀ),
84.03 (C-2ꢀ), 80.52 (C-3ꢀ), 78.38 (OCHR), 65.45, 65.22 (C-5ꢀ), 51.97 (OCH3), 48.16, 48.09

2060
W. Z. CHEN AND Y. F. ZHAO
(Cmenthyl), 42.44, 42.14 (C-α, Cmenthyl), 33.66 (Cmenthyl), 31.13 (Cmenthyl), 26.79 (>CCH3),
25.31 (>CCH3), 24.92 (Cmenthyl), 22.45 (Cmenthyl), 21.67 (CH3), 20.68 (CH3), 15.33, 15.27
(CH3). HRMS (ESI) calcd for C25H40N3O10PNa [M +Na]: 596.2349, found 596.2350.
Alanine ester menthyl phosphoramidates of 2ꢁ,3ꢁ-isopropylideneuridine
(4b). Yield 450 mg (76.7%). IR (film) v: 3224, 1742, 1698, 1630, 1213, 1066, 1004
1
cm−1.31P NMR (CDCl3, 202 MHz): δ 7.05, 6.39. H NMR (CDCl3, 500 MHz): δ 8.98,
8.92 (br, br, 1H, H-3), 7.44, 7.39 (d, d, 1H, J = 8.0 Hz, H-6), 5.82, 5.76 (br, br, 1H, H-1ꢀ),
5.74 (d, 1H, J = 7.0 Hz, H-5), 4.86∼4.91 (m, 2H, H-2ꢀ, H-3ꢀ), 4.22∼4.35 (m, 1H, OCHR),
4.16∼4.25 (m, 3H, H-5ꢀ, H-4ꢀ), 3.90∼3.98 (m, 1H, H-α), 3.74, 3.73 (s, 3H, OCH3)7∼3.51
(m, 1H, P-NH), 2.19∼2.24 (m, 1H, Hmenthyl), 2.01∼2.12 (m, 1H, Hmenthyl), 1.73∼1.78
(m, 2H, Hmenthyl), 1.64∼1.67 (m, 2H, Hmenthyl), 1.58 (s, 3H, CH3), 1.40∼1.48 (m, 1H,
Hmenthyl), 1.39, 1.38 (d, d, 3H, J = 4.0 Hz, H-β), 1.35 (s, 3H, >CCH3), 1.23-1.33 (m, 1H,
Hmenthyl), 0.96∼1.15 (m, 1H, Hmenthyl), 0.91 (t, 6H, J = 6.0 Hz, 2CH3), 0.81 (d, 3H, J = 7.0
Hz, CH3).13C NMR (CDCl3, 125 MHz): δ 174.19 (COOMe), 163.57 (C-4), 150.13 (C-2),
141.71 (C-6), 114.35 (>CMe2), 102.49-5), 93.35 (C-1ꢀ), 85.45 (C-4ꢀ), 84.18 (C-2ꢀ), 80.64
(C-3ꢀ), 78.75 (OCHR), 65.41 (C-5ꢀ), 52.19 (OCH3), 49.28, 48.88 (Cmenthyl), 42.34 (C-α),
33.78 (Cmenthyl), 31.28 (Cmenthyl), 26.91 (>CCH3), 25.46 (>CCH3), 25.04 (Cmenthyl), 22.60
(Cmenthyl), 21.79 (CH3),20.80 (CH3), 15.49 (CH3). HRMS (ESI) calcd for C26H43N3O10P:
588.2686, found 588.2688.
Valine ester menthyl phosphoramidates of 2ꢁ,3ꢁ-isopropylideneuridine
(4c). Yield 364 mg (59.2%). IR (film) v: 3237, 1734, 1694, 1622, 1214, 1063, 998 cm−1
.
31P NMR (CDCl3, 202 MHz): δ 8.02, 7.78. 1H NMR (CDCl3, 500 MHz): δ 9.02, 8.42 (br,
br, 1H, H-3), 7.45, 7.38 (d, d, 1H, J = 8.0 Hz, H-6), 5.84, 5.77 (d, d, 1H, J = 3.0 Hz, H-1ꢀ),
5.76, 5.73 (d, d, 1H, J = 8.0 Hz, H-5), 4.81–4.90 (m, 2H, H-2ꢀ, H-3ꢀ), 4.31–4.34 (m, 1H,
OCHR), 4.12–4.25 (m, 3H, H-4ꢀ, H-5ꢀ), 3.72 (s, 4H, H-α, OCH3), 3.31, 3.37 (t, t, 1H, J =
10.0 Hz, P-NH), 2.19–2.24 (m, 1H, H-β), 1.99–2.11 (m, 2H, Hmenthyl), 1.71–1.81 (m, 1H,
Hmenthyl), 1.64–1.66 (m, 2H, Hmenthyl), 1.58 (s, 3H, >CCH3), 1.38–1.45 (m, 1H, Hmenthyl),
1.35 (s, 3H, >CCH3), 1.24–1.33 (m, 1H, Hmenthyl), 0.97–1.15 (m, 2H, Hmenthyl), 0.95 (t, 3H,
J = 7.0 Hz, CH3), 0.91, 0.88 (d, d, 9H, J = 7.0 Hz, 3CH3), 0.81 (d, 3H, J = 7.0 Hz, CH3).
13C NMR (CDCl3, 125 MHz): δ 173.34 (COOMe), 163.50 (C-4), 150.03 (C-2), 141.59,
141.18 (C-6), 114.38 (>CMe2), 102.49 (C-5), 93.28, 92.62 (C-1ꢀ), 85.08 (C-4ꢀ), 84.62,
84.13 (C-2ꢀ), 80.69, 80.44 (C-3ꢀ), 78.65 (OCHR), 65.45 (C-5ꢀ), 59.71, 59.44 (OCH3), 51.85
(C-α), 48.31 (Cmenthyl), 42.46 (Cmenthyl), 33.80 (Cmenthyl), 32.10 (C-β), 31.25 (Cmenthyl),
26.91 (>CCH3), 25.38 (>CCH3), 25.02 (Cmenthyl), 22.57 (Cmenthyl), 21.79 (CH3), 20.82
(CH3), 18.79 (C-γ ), 17.38 (C-γ ꢀ), 15.44 (CH3). HRMS (ESI) calcd for C28H47N3O10P:
616.2999, found 616.2997.
Leucine ester menthyl phosphoramidates of 2ꢁ,3ꢁ-isopropylideneuridine
(4d). Yield 562.4 mg (89.41%). IR (film) v: 3226, 1730, 1697, 1626, 1213, 1060, 999
1
cm−1
.
31P NMR (CDCl3, 202 MHz): δ 7.41, 6.80. H NMR (CDCl3, 500 MHz): δ 9.00,
8.98 (br, br, 1H, H-3), 7.46, 7.38 (d, d, 1H, J = 8.0 Hz, H-6), 5.85, 576 (d, d, 1H, J = 2.5 Hz,
H-1ꢀ), 5.75, 5.73 (d, d, 1H, J = 8.0 Hz, H-5), 4.82–4.91 (m, 2H, H-3ꢀ, H-4ꢀ), 4.30–4.35 (m,
1H, OCHR), 4.16–4.23 (m, 3H, H-4ꢀ, H-5ꢀ), 3.82–3.90 (m, 1H, H-α), 3.72 (s, 3H, OCH3),
3.44, 3.33 (t, t, 1H, J = 10.0 Hz, P-NH), 2.05–2.22 (m, 2H, H-β), 1.693–1.771 (m, 1H, H-γ ,
Hmenthyl), 1.66, 1.64 (br, 2H, Hmenthyl), 1.58, 1.57 (s, s, 3H, >CCH3), 1.40–1.51 (m, 1H,
Hmenthyl), 1.35 (s, 3H, >CCH3), 1.26–1.32 (m, 2H, Hmenthyl), 1.04–1.14 (m, 1H, Hmenthyl),
0.98–1.16 (m, 4H, Hmenthyl), 0.89–0.98 (m, 12H, 4CH3), 0.81 (t, 3H, J = 7.0 Hz, CH3). 13
C
NMR (CDCl3, 125 MHz): δ 174.41 (COOMe), 163.08 (C-4), 150.03 (C-2), 141.62 (C-6),
114.62 (>CMe2), 102.71 (C-5), 93.52 (C-1ꢀ), 85.17 (C-4ꢀ), 84.32 (C-2ꢀ), 80.78 (C-3ꢀ), 78.96

AMINO ACID METHYL ESTER 5ꢁ-PHOSPHOAMIDATES
2061
(OCHR), 65.48 (C-5ꢀ), 52.87, 52.16 (OCH3), 48.50 (Cmenthyl), 43.94 (C-α), 42.70 (Cmenthyl
,
C-β), 33.95 (Cmenthyl), 31.47 (Cmenthyl), 27.11 (>CCH3), 25.63 (>CCH3), 25.19 (Cmenthyl),
24.46 (C-γ ), 22.74 (Cmenthyl, CH3), 21.96 (CH3), 20.99 (CH3), 15.61 (CH3). HRMS (ESI)
calcd for C29H49N3O10P: 630.3156, found 630.3160.
Serine ester menthyl phosphoramidates of 2ꢁ,3ꢁ-isopropylideneuridine
(4e). Yield 384 mg (63.7%). IR (film) v: 3256, 1750, 1693, 1630, 1216, 1070, 1012 cm−1
.
31P NMR (CDCl3, 202 MHz): δ 7.75, 6.72. 1H NMR (CDCl3, 500 MHz): δ 9.34 (br, 1H,
H-3), 7.31, 7.27 (d, d, 1H, J = 8.0 Hz, H-6), 5.70, 5.69 (d, d, 1H, J = 8.0 Hz, H-5),
5.60, 5.56 (d, d, 1H, J = 2.0 Hz, H-1ꢀ), 5.04, 5.00 (dd, dd, 1H, J = 6.5, 2.0 Hz, H-2ꢀ),
4.90 (dd, 1H, J = 6.5, 3.5 Hz, H-3ꢀ), 4.06–4.33 (m, 5H, H-4ꢀ, H-5ꢀ, H-β), 4.10 (q, 1H,
J = 7.0 Hz, H-α), 3.89–3.98 (br, 2H, P-NH, OCHR), 3.73 (s, 3H, OCH3), 3.46 (br, 1H,
-OH), 2.18–2.20 (m, 1H, Hmenthyl), 1.99–2.04 (m, 1H, Hmenthyl), 1.70–1.80 (m, 2H, Hmenthyl),
1.61–1.64 (m, 2H, Hmenthyl), 1.54 (s, 3H, >CCH3), 1.37–1.44 (m, 1H, Hmenthyl), 1.33 (s,
3H, >CCH3), 1.21–1.26 (m, 1H, Hmenthyl), 1.02–1.12 (m, 1H, Hmenthyl), 0.88, 0.87 (d, d,
6H, J = 6.0 Hz, 2CH3), 0.78 (d, 3H, J = 7.0 Hz, CH3). 13C NMR (CDCl3, 125 MHz):
δ 171.89 (COOMe), 163.71 (C-4), 150.35 (C-2), 142.34 (C-6), 114.23 (>CMe2), 102.54
(C-5), 94.46 (C-1ꢀ), 85.42 (C-4ꢀ), 84.20 (C-2ꢀ), 80.95 (C-3ꢀ), 78.99 (OCHR), 65.48 (C-5ꢀ),
64.07 (C-β), 56.50 (OCH3), 52.38 (C-α), 48.35 (Cmenthyl), 42.34 (Cmenthyl), 33.85 (Cmenthyl),
31.33 (Cmenthyl), 26.96 (>CCH3), 25.46 (>CCH3), 25.09 (Cmenthyl), 22.62 (Cmenthyl), 21.87
(CH3), 20.87 (CH3), 15.49 (CH3). ESI-MS: 604 (M+H)+, 626 (M+Na)+. HRMS (ESI)
calcd for C26H43N3O11P: 604.2635, found 604.2632.
Aspartic acid ester menthyl phosphoramidates of 2ꢁ,3ꢁ-isopropylidene-
uridine (4f). Yield 440 mg (68.2%). IR (film) v: 3430, 1693, 1216, 1069, 1011 cm−1 31P
.
NMR (CDCl3, 202 MHz): δ 7.60, 6.89; 1H NMR (CDCl3, 500 MHz): δ 9.31, 9.24 (br, br,
1H, H-3), 7.41, 7.37 (d, d, 1H, J = 7.5 Hz, H-6), 5.59–5.76 (m, 2H, H-1ꢀ, H-5), 4.85–5.06
(m, 2H, H-2ꢀ, H-3ꢀ), 4.28–4.34 (m, 1H, H-4ꢀ), 4.13–4.26 (m, 2H, H-5ꢀ), 3.78–3.94 (m, 3H,
H-α, P-NH, OCHR), 3.75 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 2.72–3.12 (m, 2H, H-β),
2.21–2.27 (m, 1H, Hmenthyl), 2.01–2.13 (m, 1H, Hmenthyl), 1.651–1.676 (m, 2H, Hmenthyl),
1.58, 1.57 (s, 3H, CH3), 1.40–1.48 (m, 1H, Hmenthyl), 1.37, 1.35 (s, 3H, CH3), 1.22–1.29
(m, 2H, Hmenthyl), 0.96–1.15 (m, 2H, Hmenthyl), 0.92 (d, 6H, J = 6.5 Hz, 2CH3), 0.82 (d,
3H, J = 7.0 Hz, CH3); 13C NMR (CDCl3, 125 MHz): δ 171.06 (2COOMe), 163.08 (C-4),
150.01 (C-2), 141.61 (C-6), 114.34 (>CMe2), 102.54 (C-5), 93.54 (C-1ꢀ), 85.34 (C-4ꢀ),
84.56 (C-2ꢀ), 80.39 (C-3ꢀ), 72.13 (OCHMe2), 65.53 (C-5ꢀ), 52.79, 52.04 (OCH3), 50.97
(C-α), 48.23 (Cmenthyl), 42.60 (Cmenthyl), 38.46 (C-β), 34.00 (Cmenthyl), 31.48 (Cmenthyl),
25.66, 25.31 (Cmenthyl), 22.47 (Cmenthyl), 21.75 (CH3), 20.75 (CH3), 15.37 (CH3). HRMS
(ESI) calcd for C28H44N3O12PNa [M+Na]: 668.2560, found 668.2556.
Phenylalanine ester menthyl phosphoramidates of 2ꢁ,3ꢁ-isopropylidene-
uridine (4g). Yield 503.8 mg (75.9%). IR (film) v: 3222, 1742, 1693, 1630, 1455, 1214,
1063, 998 cm−1 31P NMR (CDCl3, 202 MHz): δ 7.50, 6.81. 1H NMR (CDCl3, 500 MHz):
.
δ 8.81, 8.68 (br, br, 1H, H-3), 7.40, 7.33 (d, d, 1H, J = 7.5 Hz, H-6), 7.22–7.31 (m, 5H,
HPh), 5.80, 5.75 (br, br, 1H, H-1ꢀ), 5.686 (d, 1H, J = 7.0 Hz, H-5), 4.767–4.861 (m, 2H,
H-2ꢀ, H-3ꢀ), 4.284–3.947 (m, 5H, H-4ꢀ, H-5ꢀ, H-α, OCHR), 3.70, 3.67 (s, s, 3H, OCH3),
3.03 (d, 2H, J = 6.0 Hz, H-β), 3.31 (t, 1H, J = 10.0 Hz, P-NH), 1.98–2.24 (m, 2H,
Hmenthyl), 1.70–1.79 (m, 2H, Hmenthyl), 1.64–1.66 (m, 2H, Hmenthyl), 1.57 (s, 3H, >CCH3),
1.36–1.47 (m, 1H, Hmenthyl),1.35 (s, 3H, >CCH3), 1.22–1.30 (m, 1H, Hmenthyl), 1.07–1.15
(m, 1H, Hmenthyl), 0.905 (d, 6H, J = 6.0 Hz, 2CH3), 0.784 (d, 3H, J = 7.0 Hz, CH3).
13C NMR (CDCl3, 125 MHz): δ 172.93 (COOMe), 163.37 (C-4), 150.03 (C-2), 141.52
(C-6), 135.77 (CPh), 129.32 (CPh), 128.40 (CPh), 126.94 (CPh), 114.48 (>CMe2), 102.56

2062
W. Z. CHEN AND Y. F. ZHAO
(C-5), 93.23 (C-1ꢀ), 84.96 (C-4ꢀ), 84.20 (C-2ꢀ), 80.64 (C-3ꢀ), 78.84 (OCHR), 65.33 (C-5ꢀ),
55.36 (OCH3), 52.09 (C-α), 48.35 (Cmenthyl), 42.58 (Cmenthyl), 40.45 (C-β), 33.87 (Cmenthyl),
31.33 (Cmenthyl), 27.01 (>CCH3), 25.53 (>CCH3), 25.17 (Cmenthyl), 22.67 (Cmenthyl), 21.87
(CH3), 20.92 (CH3), 15.56 (CH3); HRMS (ESI) calcd for C32H47N3O10P: 664.2999, found
664.3004.
Tyrosine ester menthyl phosphoramidates of 2ꢁ,3ꢁ-isopropylideneuridine
(4h). Yield 442.1 mg (65.1%). IR (film) v: 3253, 1710, 1692, 1615, 1517, 1455, 1216,
1067, 1017 cm−1.31P NMR (CDCl3, 202 MHz): δ 7.56, 6.60; 1H NMR (CDCl3, 500 MHz):
δ 9.79, 9.64 (br, br, 1H, H-3), 7.89–7.73 (br, 1H, -OH), 7.34, 7.30 (d, d, 1H, J = 8.0 Hz, H-6),
6.94–6.97 (m, 2H, HPh), 6.75–6.78 (m, 2H, HPh), 5.72, 5.67 (d, 1H, J = 2.0 Hz, H-1ꢀ), 5.66,
5.65 (d, d, 1H, J = 8.0 Hz, H-5), 4.917–4.96 (m, 2H, H-2ꢀ, H-3ꢀ), 4.87–4.88 (m, 1H, H-4ꢀ),
4.69–4.78 (m, 3H, H-5ꢀ, OCHR), 3.90–3.99 (m, 1H, H-α), 3.71, 3.70 (s, s, 3H, OCH3),
3.57 (t, 1H, J = 10.5 Hz, P-NH), 2.92–2.96 (m, 2H, H-β), 2.01–2.21 (m, 2H, Hmenthyl),
1.69–1.78 (m, 2H, Hmenthyl), 1.61–1.69 (m, 2H, Hmenthyl), 1.55 (s, 3H, CH3), 1.39–1.47 (m,
1H, Hmenthyl), 1.34, 1.33 (s, s, 3H, CH3), 1.22–1.30 (m, 1H, Hmenthyl), 0.95–1.14 (m, 1H,
Hmenthyl), 0.90 (d, 3H, J = 7.0 Hz, CH3), 0.87 (d, 3H, J = 7.0 Hz, CH3), 0.79, 0.78 (d, d,
3H, J = 7.0 Hz, CH3); 13C NMR (CDCl3, 125 MHz): δ 173.42 (COOMe), 163.88 (C-4),
155.88 (PhOH), 150.79 (C-2), 142.00, 141.69 (C-6), 130.41 (CPh), 126.82 (CPh), 126.43
(CPh), 115.66 (CPh), 114.45 (>CMe2), 102.44 (C-5), 93.96, 93.44 (C-1ꢀ), 85.03 (C-4ꢀ),
84.23 (C-2ꢀ), 80.69 (C-3ꢀ), 79.28 (OCHR), 65.38 (C-5ꢀ), 55.97 (OCH3), 52.21 (C-α), 48.38
(Cmenthyl), 42.51 (Cmenthyl), 39.52 (C-β), 33.87 (Cmenthyl), 31.33 (Cmenthyl), 27.01 (>CCH3),
25.50 (>CCH3), 25.12 (Cmenthyl), 22.64 (Cmenthyl), 21.87 (CH3), 20.92 (CH3), 15.56 (CH3).
HRMS (ESI) calcd for C32H47N3O11P: 680.2948, found 680.2942.
Histidine ester menthyl phosphoramidates of 2ꢁ,3ꢁ-isopropylideneuri-
dine (4i). Yield 465.6 mg (71.3%). IR (film) v: 3246, 1705, 1693, 1610, 1217, 1054,
999 cm−1 31P NMR (CDCl3, 202 MHz): δ 7.25, 6.40; 1H NMR (CDCl3, 500 MHz): δ 7.58
.
(s, 1H, Himidazole), 7.41 (d, 1H, J = 8.0 Hz, H-6), 6.81, 6.77 (s, s, 1H, Himidazole), 5.70 (br, 1H,
H-1ꢀ), 5.65 (d, 1H, J = 8.0 Hz, H-5), 4.86–4.93 (m, 2H, H-2ꢀ, H-3ꢀ), 4.25–4.31 (m, 2H, H-4ꢀ,
OCHR), 4.14 (br, 3H, H-5ꢀ, P-NH), 4.05 (t, 1H, J = 5.5 Hz, H-α), 3.69 (s, 3H, OCH3),
3.11, 3.06 (d, d, 2H, J = 3.5 Hz, H-β), 2.15-2.18 (m, 1H, Hmenthyl), 2.03–2.12 (m, 1H,
Hmenthyl), 1.62–1.64 (m, 2H, Hmenthyl), 1.56 (s, 3H, >CCH3), 1.37–1.47 (m, 1H, Hmenthyl),
1.34 (s, 3H, >CCH3), 1.26–1.30 (m, 2H, Hmenthyl), 0.93–1.09 (m, 2H, Hmenthyl), 0.88, 0.87
(d, d, 6H, J = 6.5 Hz, 2CH3), 0.78 (d, 3H, J = 7.0 Hz, CH3); 13C NMR (CDCl3, 125
MHz): δ 173.25 (COOMe), 164.08 (C-4), 150.54 (C-2), 141.93 (C-6), 135.41 (Cimidazole),
133.16 (Cimidazole), 119.58 (Cimidazole), 114.18 (>CMe2), 102.49 (C-5), 93.88 (C-1ꢀ), 85.25
(C-4ꢀ), 84.35 (C-2ꢀ), 80.90 (C-3ꢀ), 78.96 (OCHR), 65.33 (C-5ꢀ), 54.83 (OCH3), 52.19 (C-α),
48.43 (Cmenthyl), 42.41 (Cmenthyl), 35.82 (Cmenthyl), 31.33 (C-β, Cmenthyl), 27.01 (>CCH3),
25.46 (>CCH3), 25.12 (Cmenthyl), 22.62 (Cmenthyl), 21.87 (CH3), 20.90 (CH3), 15.51 (CH3).
HRMS (ESI) calcd for C29H45N5O10P: 654.2904, found 654.2899.
Methionine ester menthyl phosphoramidates of 2ꢁ,3ꢁ-isopropylidene-
uridine (4j). Yield 472.3 mg (73.1%). IR (film) v: 3216, 1722, 1694, 1625, 1216, 1063,
1
1011 cm−1.31P NMR (CDCl3, 202 MHz): δ 7.49, 6.68; H NMR (CDCl3, 500 MHz): δ
8.91 (br, 1H, H-3), 7.36, 7.28 (d, d, 1H, J = 8.0 Hz, H-6), 5.63–5.74 (m, 2H, H-1ꢀ, H-5),
4.77–4.89 (m, 2H, H-2ꢀ, H-3ꢀ), 4.24–4.27 (m, 1H, H-4ꢀ), 4.06–4.21 (m, 3H, H-5ꢀ, OCHR),
3.91–4.00 (m, 1H, H-α), 3.67 (s, 3H, OCH3), 3.46–3.55 (m, 1H, P-NH), 2.49, 2.48 (d, 2H,
J = 3.0 Hz, H-γ ), 2.22, 2.15 (d, 2H, J = 10.5 Hz, H-β), 2.02 (s, 3H, SCH3), 1.94–2.00
(m, 1H, Hmenthyl), 1.80–1.91 (m, 1H, Hmenthyl), 1.67–1.77 (m, 2H, Hmenthyl), 1.54–1.63 (m,
2H, Hmenthyl), 1.51 (s, 3H, >CCH3), 1.33–1.43 (m, 1H, Hmenthyl), 1.28 (s, 3H, >CCH3),

AMINO ACID METHYL ESTER 5ꢁ-PHOSPHOAMIDATES
2063
1.19–1.23 (m, 1H, Hmenthyl), 1.01–1.06 (m, 1H, Hmenthyl), 0.84 (s, 6H, 2CH3), 0.74 (s, 3H,
CH3); 13C NMR (CDCl3, 125 MHz): δ 173.32 (COOMe), 163.50 (C-4), 150.11 (C-2),
141.70, 141.38 (C-6), 114.26 (>CMe2), 102.44 (C-5), 93.44, 92.94 (C-1ꢀ), 85.01, 84.74
(C-4ꢀ), 84.11 (C-2ꢀ), 80.69, 80.47 (C-3ꢀ), 78.72 (OCHR), 65.41 (C-5ꢀ), 53.20 (C-α), 52.19
(OCH3), 48.23 (Cmenthyl), 42.36 (Cmenthyl), 33.68, 33.44 (C-β), 31.20 (Cmenthyl), 29.44 (C-
γ ), 26.86 (>CCH3), 25.31 (>CCH3), 25.00 (Cmenthyl), 22.47 (Cmenthyl), 21.75 (CH3), 20.75
(CH3), 15.37 (CH3), 15.08 (SCH3). HRMS (ESI) calcd for C28H47N3O10PS: 648.2720,
found 648.2717.
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