Synthesis and hydrolysis of a phenylalanyl adenylate pentacoordinated phosphorane

Article abstract of DOI:10.1016/S0045-2068(03)00023-3

Amino acid-nucleotide conjugates have important biological functions and therapeutic applications. For example, aminoacyl adenylates are key intermediates in aminoacyl tRNA synthetase reactions. They may also be involved in the prebiotic synthesis of polypeptides. Finally, various amino acid carbomethoxy aryl phosphoramidates of nucleotide prodrugs may be activated through a mechanism involving a pentacoordinated phosphorane intermediates. In order to understand better the chemistry of these compounds, a phenylalanyl adenylate pentacoodinated phosphorane has been synthesized in 72% yield and its decomposition in aqueous solution studied. Hydrolysis gave 2′,3′-O-isopropylidene adenosine 5′-monophosphate, 2′,3′-O-isopropylidene adenosine, and phenylalanine. The results provide model chemistry for the enzymatic degradation mechanism of antiviral aryl amino acid phosphodiester amidates in cells, which leads to their activation.

Full text of DOI:10.1016/S0045-2068(03)00023-3

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 31 (2003) 122–128
www.elsevier.com/locate/bioorg
Synthesis and hydrolysis of a
phenylalanyl adenylate pentacoordinated
phosphorane
Hua Fu,a,* Bo Han,a Yu-Fen Zhao,a Guang-Zhong Tu,b
Li Xu,b Qiang Lu,b Jing-Zun Wang,b and Hong-Zhan Xiaob
a
Department of Chemistry, Bioorganic Phosphorus Chemistry Lab of Education Ministry,
School of Life Sciences and Engineering, Tsinghua University, Beijing 100084, PeopleÕs Republic of China
b
Beijing Institute of Microchemistry, Beijing 100091, PeopleÕs Republic of China
Received 3 June 2002
Abstract
Amino acid-nucleotide conjugates have important biological functions and therapeutic ap-
plications. For example, aminoacyl adenylates are key intermediates in aminoacyl tRNA syn-
thetase reactions. They may also be involved in the prebiotic synthesis of polypeptides.
Finally, various amino acid carbomethoxy aryl phosphoramidates of nucleotide prodrugs
may be activated through a mechanism involving a pentacoordinated phosphorane intermedi-
ates. In order to understand better the chemistry of these compounds, a phenylalanyl adenyl-
ate pentacoodinated phosphorane has been synthesized in 72% yield and its decomposition in
aqueous solution studied. Hydrolysis gave 20,30-O-isopropylidene adenosine 50-monophos-
phate, 20,30-O-isopropylidene adenosine, and phenylalanine. The results provide model chem-
istry for the enzymatic degradation mechanism of antiviral aryl amino acid phosphodiester
amidates in cells, which leads to their activation.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Phenylalanyl adenylate pentacoordinated phosphorane; Nucleotide prodrugs; Hydrolysis of
phosphodiester amidates
* Corresponding author. Fax: +86-10-62781695.
E-mail address: fuhua@mail.tsinghua.edu.cn (H. Fu).
0045-2068/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0045-2068(03)00023-3

H. Fu et al. / Bioorganic Chemistry 31 (2003) 122–128
123
Compounds in which amino acids are linked to nucleotides are found in nature
and have significant biological and medical importance. Aminoacyl adenylates are
enzymatic reaction intermediates in aminoacyl tRNA synthetase reactions resulting
from the reaction of amino acids with ATP in the biosynthesis of proteins [1]. They
have also been implicated as intermediates in the prebiotic synthesis of polypeptides
[2]. In our previous research, N,O-bis(trimethylsilyl)-a-amino acids, but not b-amino
acids, could oligomerize into polypeptides in the presence of o-phenylene phospho-
rochloridate. The mechanism preceded through a five-membered cyclic pentacoordi-
nated phosphoranes containing the a-amino acid residue and was a key step in the
oligomerization reaction. This observation suggested that phosphates could prefer-
entially choose a-amino acids in the prebiotic synthesis of polypeptides [3]. In addi-
tion, the activation mechanism for various amino acid carbomethoxy ester aryl
phosphoramidate nucleotide prodrugs (1) in cells to form 50-monophosphate (6)
and nucleoside (7) most likely involves pentacoordinated phosphoranes (3) as shown
in Scheme 1 [4–6]. Amino acid phosphoramidates of nucleosides have shown promise
as potent antiviral agents, because in some cases they have exhibited enhanced antiv-
iral activity and reduced cytotoxicity when compared to the parent nucleosides [7–9].
For these reasons it is very important to synthesize a pentacoordinated phosphorane
containing amino acid and nucleoside components and study the hydrolysis path-
way.
1. Materials and methods
All chemicals were purchased from Beijing Chemical Reagent in China. Proton,
carbon, and phosphorus nuclear magnetic resonance (1H, 13C, and 31P NMR) spec-
tra were recorded on a Bruker AC spectrometer operating at 500, 125, and 81 MHz,
respectively. Chemical shifts for 31P NMR spectra are indicated in parts per million
relative to an external 85% phosphoric acid standard. Field desorption mass spec-
trum (FDMS) was obtained on a Finnigan MAT 90 mass spectrometer. LC-ESI
mass spectra were recorded on Bruker Esquire-LC electrospray ionization mass
spectrometry.
Scheme 1. Proposed amino acid decomposition pathway of antiviral aryl amino acid phosphodiester am-
idates [4–6].

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H. Fu et al. / Bioorganic Chemistry 31 (2003) 122–128
1.1. Synthesis of 20,30-O-isopropylidene-50-O-(trimethylsilyl)adenosine (10)
Compound 10 was obtained from the reaction of 20,30-O-isopropylidene adenosine
and an excess of hexamethyldisilazane (HMDS, 1.5 eq) in dry CH2Cl2 by refluxing
under a nitrogen atmosphere until a clear solution appeared (about 2.5 h). The excess
1
HMDS and the solvent were removed by distillation leaving 10. An H NMR spec-
trum showed that the purity was greater than 95%. The product was not purified fur-
1
ther. H NMR (500 MHz, CDCl3) of 10: d 0.18 (s, 9H, SiMe3), 1.31, 1.53 (ss, 6H,
H12, H13), 3.51–3.58 (m, 2H, H50), 4.21 (m, 1H, H40), 4.96 (d, 1H, H30), 5.33 (m,
1H, H20), 6.12 (d, 1H, H10), 7.32 (s, 2H, H10), 8.16 (s, 1H, H8), 8.33 (s, 1H, H2).
1.2. Procedure for the preparation of 11
To a stirring solution of 2,2,2-trichloro-1,3,2-benzodioxaphosphole (8) (1.23 g,
5 mmol) in dry CH2Cl2 (10 mL) at room temperature under a nitrogen atmosphere
was added dropwise a solution of N,O-bis(trimethylsilyl)phenylalanine (5 mmol) in
dry CH2Cl2 (5 mL). After 10 min, 8 was almost quantitatively converted into 9, and
a solution of 20,30-O-isopropylidene-50-O-(trimethylsilyl)adenosine (10) (1.89 g,
5 mmol) was added dropwise to the resulting solution. The reaction was complete
in 15 min. Subsequently, solvent was removed by evaporation in vacuo, and the tar-
get product 11 was obtained as a white solid after crystallization from CH2Cl2 and
ethyl ether in 72% yield (2.2 g). 31P NMR (81 MHz, CH2Cl2): d )41.64, )41.92 ppm
1
(integral area 1:1); H NMR (500 MHz, CDCl3): d 1.07–1.56 (m, 6H, H12, H13),
2
3.22–3.36 (m, 2H, H23), 3.75 (m, 1H, H20, JP–NH ¼ 8:2 Hz), 3.91 (m, 1H, H21),
3.98 (m, 1H, H40), 4.18 (m, 1H, H30), 4.36 (m, 1H, H20), 4.48 (m, 2H, H50), 6.12
(m, 1H, H10), 6.52–7.20 (m, 9H, H15, 16, 17, 18, 25, 26, 27, 28, 29), 7.42 (d, 2H,
H10), 8.82 (d, 1H, H8), 9.36 (d, 1H, H2); 13C NMR (125 MHz, CDCl3): d 24.59,
27.00 (C12, C13), 37.05 (C23), 56.21 (C21), 68.45 (C50), 81.60 (C20), 85.26 (C30),
85.88 (C40), 91.93 (C10), 109.94 (C15), 111.56 (C18), 114.49 (C11), 117.90 (C5),
120.89 (C17), 123.51 (C16), 127.15 (C27), 128.29 (C26, 28), 131.26 (25, 29),
135.45 (C8), 142.04 (C24), 142.53 (C19), 143.96 (C14), 144.46 (C4), 148.50 (C2),
151.07 (C6), 169.06 (C22, JP–C ¼ 17:8 Hz); Positive ion FDMS Mþ m=z 608.
2
It was not possible to obtain a melting point because 11 was labile at high
temperature.

H. Fu et al. / Bioorganic Chemistry 31 (2003) 122–128
125
1.3. Hydrolysis of compound 11
Compound 11 (12.2 mg, 0.02 mmol) was dissolved in acetone (0.6 mL), and the so-
lution was transferred into an NMR tube. Water (2 eq) was added to the solution
and the tube was shaken. Subsequently, the hydrolysis of 11 was monitored by
81 MHz 31P NMR spectroscopy at room temperature.
1.4. LC-ESI MS analytical conditions
LC-ESI MS analysis of the hydrolysis products of 11 was carried on Varian
VISTA 5500 liquid chromatograph with a reversed-phase C18 column (ZORBAX
SB-C18) and Bruker Esquire-LC-ESI mass spectrometer. Elution was accomplished
with 20% acetonitrile and 0.1% acetic acid aqueous solution at a flow rate of 0.8 mL/
min.
2. Results and discussion
The phenylalanyl adenylate pentacoordinated phosphorane (11) was synthesized
by the sequential reaction of 2,2,2-trichloro-1,3,2-benzodioxaphosphole [10] (8) with
N,O-bis(trimethylsilyl) phenylalanine [11] and 20,30-isopropylidene-50-O-(trimethylsi-
lyl)adenosine (10). The synthetic pathway is shown in Scheme 2.
There are two advantages using 20,30-O-isopropylidene-50-O-(trimethylsilyl)-aden-
osine (10) instead of 20,30-O-isopropylidene adenosine/triethylamine. First, the isola-
tion of the target product is easier by recrystallization in the absence of triethylamine
hydrochloride. Second, nucleophilic attack of the 50-hydroxyl of 20,30-O-isopropylid-
ene adenosine to the carbonyl of the mixed anhydride is avoided. It is interesting to
note that the reaction occurred on 50-O-(trimethylsilyl)oxygen of 20,30-O-isopropylid-
ene-50-O-(trimethylsilyl)adenosine rather than on the free amino group of adenine.
31P NMR integral areas of 11 showed that the product was a 1:1 mixture of diaste-
reomers.
Scheme 2. Synthetic pathway of phenylalanyl adenylate pentacoordinated phosphorane (11).

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H. Fu et al. / Bioorganic Chemistry 31 (2003) 122–128
The reaction of 11 with water in acetone was monitored by 31P NMR spectros-
copy at room temperature (Fig. 1). As the reaction progressed, the pair of peaks
at )41.64 and )41.92 ppm, corresponding to the two diastereomers of 11 gradually
decreased. At the same time, the peaks at )3.74 and )3.52 ppm, corresponding to
phosphodiester (15) and phosphomonoester (16), respectively, increased. A small
signal at 13.56 ppm, corresponding to the cyclicphosphotriester [12] (14), also ap-
peared as shown in Fig. 1. After 12 h, 11 was completely converted to the final prod-
ucts 15–18, catechol and nucleoside monophosphate (19). The structures of 15–18
were verified by LC-ESI-MS/MS. The peaks at 3.19, 3.62, 6.33, and 9.45 min
(Fig. 2) correspond to compounds 17, 16, 18, and 15, respectively, whose protonated
molecules ½M þ Hꢀþ are m=z 166, 191, 308, and 480 in ESI-MS. Based on these ob-
Fig. 1. The 31P NMR spectra monitoring the hydrolysis reaction for phenylalanyl adenylate pentacoordi-
nated phosphorane (11). The signals corresponding to the diastereomers of 11 ()41.64 and )41.92 ppm)
are decreasing in intensity while the signals corresponding to 15, 16, and 19 (from )3.74 to )3.52 ppm)
are increasing in intensity.
Fig. 2. A chromatogram of the hydrolysis products for phenylalanyl adenylate pentacoordinated phos-
phorane (11). The peaks at 3.19, 3.62, 6.33, and 9.45 min, corresponding to 17, 16, 18, and 15, were
identified by ESI-MS.

H. Fu et al. / Bioorganic Chemistry 31 (2003) 122–128
127
servations, a hydrolysis pathway for 11 is proposed in Scheme 3. Nucleophilic attack
of water on the phosphorus of 11 leads to the cleavage of the P–N bond or the P–O
bond of the mixed anhydride bond to form intermediate 12 or 13. Futher hydrolysis
of 12 results in 14 with the loss of 17 while futher hydrolysis of 13 leads to 16 with the
loss of 17 and adenosine (18). Subsequently, 14 was hydrolyzed into the phosphodi-
ester (15), which, in turn, could produce catechol and 19. The compounds corre-
spond to two peaks within 3 min in Fig. 2. Unfortunately their protonated
molecules ½M þ Hꢀþ were not determined because their sensitivity is low in positive
ion ESI-MS.
The structure of compound 11 is similar to that of proposed intermediate 3 in
Scheme 1, and the products of hydrolysis also are in agreement with the observed
enzymatic degradation products of antiviral aryl amino acid phosphodiester ami-
dates in cells [4–6].
In summary, we have developed a mild procedure for the production of amino-
acyl adenylate pentacoordinated phosphoranes. Hydrolysis of the pentacoodinated
phosphorus compound yields the phosphodiester, phosphomonoester, nucleoside,
and amino acid. The hydrolysis pathway could provide model chemistry for
enzymatic degradation mechanism of antiviral aryl amino acid phosphodiester
amidates.
Scheme 3. Possible hydrolysis pathway for the phenylalanyl adenylate pentacoordinated phosphorane
(11).

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H. Fu et al. / Bioorganic Chemistry 31 (2003) 122–128
Acknowledgments
The work was supported by the National Natural Science Foundation of China
(No. 29902003), the National Science and Technology Committee of China, the
Chinese Education Ministry and Tsinghua University.
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