Intermolecular phosphoryl transfer of N-phosphoryl amino acids

Article abstract of DOI:10.1002/ejoc.201100234

N-Phosphoryl amino acids (NPAAs) are a novel series of N-terminal-activated amino acids that act as the energy source and phosphoryl donor in intra- and intermolecular phosphoryl transfer to form "high-energy" species, such as acetyl phosphate and aminoacyl phosphates, and in the self-assembled synthesis of polypeptides under mild aqueous conditions. In this work, the chemical reactivity of N-mono(methoxyphosphoryl)glycine as a representative was investigated in detail by using a combination of the stable-isotope-labeling (¹⁵N) technique, ³¹P NMR, ESI-MS/MS and LC-MS. The phosphoryl group of NPAAs can be transferred intermolecularly to the carboxy group of another molecule through intramolecular cyclic pentacoordinate phosphoric-amino acid anhydride intermediates. In addition to C-terminal activation by phosphate anhydride, amino acids can also be self-activated by N-phosphorylation. This information not only provides some interesting clues for understanding the active role of the phosphoryl group in living systems, but also shows that the origin of life might be attributed to the chemical evolution of N-phosphoryl amino acids. Copyright

Full text of DOI:10.1002/ejoc.201100234

FULL PAPER
DOI: 10.1002/ejoc.201100234
Intermolecular Phosphoryl Transfer of N-Phosphoryl Amino Acids
Xiang Gao,[a] Honggui Deng,[a] Guo Tang,[a] Yan Liu,[a] Pengxiang Xu,[a] and
Yufen Zhao*[a,b]
Keywords: Reaction mechanisms / Phosphorus / Amino acids / Origin of life
N-Phosphoryl amino acids (NPAAs) are a novel series of N-
terminal-activated amino acids that act as the energy source
and phosphoryl donor in intra- and intermolecular phos-
phoryl transfer to form “high-energy” species, such as acetyl
phosphate and aminoacyl phosphates, and in the self-
assembled synthesis of polypeptides under mild aqueous
conditions. In this work, the chemical reactivity of N-mono-
(methoxyphosphoryl)glycine as a representative was investi-
gated in detail by using a combination of the stable-isotope-
labeling (15N) technique, 31P NMR, ESI-MS/MS and LC-MS.
The phosphoryl group of NPAAs can be transferred intermo-
lecularly to the carboxy group of another molecule through
intramolecular cyclic pentacoordinate phosphoric–amino
acid anhydride intermediates. In addition to C-terminal acti-
vation by phosphate anhydride, amino acids can also be self-
activated by N-phosphorylation. This information not only
provides some interesting clues for understanding the active
role of the phosphoryl group in living systems, but also shows
that the origin of life might be attributed to the chemical
evolution of N-phosphoryl amino acids.
phosphoramidate bond (P–N bond) has attracted increas-
ing attention because of its fundamental roles in metabo-
Introduction
In contemporary biosynthesis, amino acids are first acti- lism and signalling.[4] In our research group, N-phosphoryl
vated by C-terminal phosphorylation to form energy-rich amino acids (NPAAs) have been investigated as model com-
aminoacyl adenylates for protein synthesis, catalysed by pounds under mild conditions.[5] In NPAAs, natural α-
aminoacyl tRNA synthetases (Scheme 1).[1] Furthermore, amino acids are activated by N-phosphorylation to a higher
amino acids can be activated to form aminoacyl phosphate energy state through the intramolecular phosphoryl-carb-
anhydrides under a variety of potentially primordial condi- oxy mixed anhydride[6] and some interesting biomimetic re-
tions.[2] Because of the prevalence of these compounds, actions have been observed, including self-assembly oligo-
there have been many investigations into the inherent nature peptide formation, ester exchange on the phosphoryl group,
of the aminoacyl phosphate anhydride as key intermedi- carboxylic ester formation and an intramolecular phos-
ate.[3] On the other hand, the biologically “high-energy” phoryl group migration from nitrogen to oxygen.[7]
Scheme 1. Two pathways for amino acid activation: the C- and N-terminal phosphorylation of natural α-amino acids.
[a] Department of Chemistry and The Key Laboratory for
Chemical Biology of Fujian Province, College of Chemistry and
Chemical Engineering, Xiamen University,
Indeed, NPAAs can be produced by the reaction of
amino acids with prebiotically available linear polyphos-
phates or trimetaphosphate[8] in aqueous solution.[9] In ad-
dition, NPAAs have been considered as mini-chemical mod-
els in a study of intramolecular catalysis[10] and for the elu-
cidation of the phosphoryl transfer mechanism in kin-
ases.[11] These studies not only have shown that the origin
Xiamen 361005, P. R. China
Fax: +86-592-2185780
E-mail: yfzhao@xmu.edu.cn
[b] The Key Laboratory for Bioorganic Phosphorus Chemistry and
Chemical Biology (Ministry of Education), Department of
Chemistry, School of Sciences, Tsinghua University,
Beijing 100084, P. R. China
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100234.
3220
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3220–3228

Phosphoryl Transfer of N-Phosphoryl Amino Acids
of life might be attributed to the chemical evolution of N-
phosphoryl amino acids, but have also provided some im-
portant clues for understanding the central role of phos-
phates in living systems.[12]
In recent decades, there has been significant progress in
the design and development of amino acid phosphoramid-
ate triesters as an effective pro-drug strategy for the intra-
cellular delivery of charged anti-viral and anti-cancer nu-
cleotide monophosphates.[13] Interestingly, in different bio-
logical media and animal species, the activation of phos-
phoramidate triester pro-dugs, mediated by carboxyester-
ase-type enzymes, has been used to form a high concentra-
tion of phosphoramidate conjugates of amino acids and nu-
cleotides having P–N bonds and free carboxylic groups.[14]
Recently, it has been demonstrated that amino acid phos-
phoramidates of nucleosides can mimic the natural triphos-
phate moiety effectively and be incorporated into growing
DNA chains under catalysis by polymerases.[15] In addition,
the thermochemistry and mechanism for the hydrolysis of
aspartic acid phosphoramidate nucleotide as a model com-
pound has been investigated by comparative theoretical cal-
culations.[16] The gas-phase chemistry of amino acid phos-
phoramidates of adenosine and N-phosphoryl amino acids
Scheme 2. “High-energy” phosphoester bond and peptide forma-
tion of N-phosphorylglycine in aqueous solution.
Results and Discussion
The reaction of N-MMP-Gly in aqueous solution was
has been explored by electrospray ionization mass spec- monitored by 31P NMR spectroscopy and LC-ESI-MS.
trometry.[17] However, to the best of our knowledge, the Within a pD range of 8.0–9.0 and at a temperature of 40 °C,
chemistry of amino acid phosphoramidates of nucleosides there was no degradation of N-MMP-Gly (0.2 m) observed
has remained relatively unexplored in spite of their poten- after a period of 8 d (Figure 1, a). In contrast, as shown
tially significant biological and pharmaceutical applica- in part b of Figure 1 at pD 4.0–5.0, N-MMP-Gly (0.2 m)
tions.[18]
decomposes to generate products 2 [δ = 9.2 ppm, N-mono-
Initially we found that the nucleotide portion of the mo- (methoxyphosphoryl)glycylglycine (N-MMP-Gly-Gly, con-
lecule has undesirable chemical complexity because of its firmed by comparison with an authentic sample, Figure S1,
multiple functional groups. The desired reaction pathways Supporting Information)], 3 (δ = 1.7 ppm, methyl phos-
need to emerge primarily from the N-phosphoryl amino phate), 4 (δ = 0.1 ppm, PO43–) and 6 (δ = –9.6 ppm, di-
acid moiety having two reactive centres, at the phosphoryl methyl pyrophosphate, confirmed by LC-MS; Figure S2,
and carbonyl groups, independent of the nucleotide group. Supporting Information). This dependence of reactivity on
Based on these considerations, it appeared more expedient mild acid conditions established the reactive species as the
to study simpler monoesters of N-phosphoryl amino acids monosodium salt of the methyl phosphonamidate with the
to retain the potentially chemically useful properties of carboxylic acid un-ionized, as seen in earlier work by Black-
amino acid phosphoramidates of nucleosides while avoiding burn and Kirby and their co-workers.[21] Detailed analysis
the complexity of the nucleoside groups. The present study of the reaction identifies an intermediate 5 with two phos-
focuses on the reactions of the disodium salt of N-mono- phorus signals at δ = 8.7 and –5.9 ppm that appears imme-
(methoxyphosphoryl)glycine (N-MMP-Gly, 1) as a typical diately, increases to a maximum after 3.5 h and then slowly
member of the general class in the context of phosphoryl disappears over 18 h. A 1:1 ratio of 5-PN/5-PC is seen by
group transfer (Scheme 2). The phosphoryl group of 31P NMR spectroscopy during the formation of 5. The sing-
NPAAs can be transferred intermolecularly to the carboxy let peak of 5-PN at δ = 8.7 ppm changes into multiple peaks
1
group of another molecule through intramolecular cyclic (JH-P = 10.7 Hz, tq) in the H-coupled 31P NMR spectrum
pentacoordinate phosphoric-amino acid anhydride interme- (Figure 2, b), and is similar to reactant 1 and N-MMP-Gly-
diates. Besides C-terminal activation by phosphate anhy- Gly 2, resulting from splitting by the methyl and methylene
drides, amino acids can also be self-activated by N-phos- groups. To establish the reaction pathway, stable-isotope-
phorylation. In parallel with the formation of polypeptides, labelled 15N-Gly was incorporated into N-MMP-Gly. Un-
“high-energy” phosphoester bonds are formed by phos- der the same reaction conditions, the 1H-decoupled 31P
phoryl transfer reactions that have been referred to as “the NMR spectrum of the reaction mixture derived from 15N-
centrepiece of biochemical processes”[19] through the break- labelled N-MMP-Gly showed a doublet at δ = 8.7 ppm
ing of the P–N bond of an N-phosphoryl amino acid under (JN-P = 35.6 Hz), indicative of a P–N bond in intermediate
mild aqueous conditions. Note that the biologically impor- 5 (Figure 2, c). The quartet (JH-P = 11.2 Hz) and singlet
tant divalent magnesium ion can catalyse the formation of observed in Figure 2, parts e and f, respectively, are as ex-
pyrophosphate, which may have acted as an ancient energy pected for the PC signal of 5. These NMR spectroscopic
carrier in place of ATP.[20]
data are also in agreement with the reported values for the
Eur. J. Org. Chem. 2011, 3220–3228
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3221

X. Gao, H. Deng, G. Tang, Y. Liu, P. Xu, Y. Zhao
FULL PAPER
structure of the mixed phosphoric-carboxylic anhydride.[2,3]
Consequently, intermediate 5 is proposed as the structure
of the phosphate anhydride of N-MMP-Gly with the for-
mation of the “high-energy” phosphoester bond
(Scheme 2). In addition, LC-ESI-MS analysis of the reac-
tion mixture after 8 d incubation at 40 °C showed the for-
mation of diglycine (G2, 13.0% yield), triglycine (G3, 1.2%
yield) and trace amounts of tetraglycine (Table 1).
Figure 2. 31P NMR analysis of the products 1, 2 and 5 formed in
the reaction of N-MMP-Gly (a,d and b,e) and stable-isotope-
labeled 15N-MMP-Gly (c,f) incubated in D2O.
7 with a synthetic sample of acetyl phosphate (Figure S3,
Supporting Information). Thus, the phosphoryl group of
N-MMP-Gly can be transferred to acetic acid to form ace-
tyl phosphate, and this can serve two different purposes,
either to transfer its phosphoryl group into the phosphate
pool or to supply its active acetyl group for the biosynthesis
of the carbon structures.[22]
The reaction of N-MMP-Gly (0.2 m) with glycine (0.2 m)
in D2O was also tracked by 31P NMR spectroscopy (Fig-
ure 3, b). Indeed, intermediate 8 is observed at –6.2 ppm,
which is consistent with values reported for aminoacyl ethyl
phosphate[4b] and shows similar kinetic behaviour. The
structure of the mixed anhydride 8 was also confirmed by
comparison with an authentic sample of glycyl methyl
phosphate (Figure S4, Supporting Information). In the re-
action of N-MMP-Gly with alanine, a carboxy-activated in-
termediate can be detected at δ = –6.1 ppm and its structure
was confirmed as alaninyl methyl phosphate 9 by compari-
son with an authentic sample (Figure S5, Supporting Infor-
mation).
It is thus evident that N-phosphoryl amino acids are not
only self-activated to produce “high-energy” phosphoric-
acetyl anhydride bonds, but also deliver reasonable yields of
oligopeptides for several N-phosphoryl amino acids under
different reaction conditions (Table 1 and Figure S6, Sup-
Figure 1. Time-dependent, stacked 31P NMR spectra for the reac-
tions of N-MMP-Gly in aqueous solution at 40 °C for 8 d. (a) N-
MMP-Gly (0.2 m) at pD 8.0–9.0; (b) N-MMP-Gly (0.2 m) at
pD 4.0–5.0.
To investigate the effect of the intermolecular phosphoryl porting Information). Variations in the chemical structure
transfer reaction, the reaction of N-MMP-Gly (0.2 m) with of the alkyl group and the concentration of N-MMP-Gly
acetic acid (0.2 m) in aqueous solution was monitored (Fig- had no significant effect on the yields of phosphates and
ure 3, a). This showed that in addition to product 5, acetyl peptides (entries 1–3). However, the product yields depend
phosphate 7 was detected (–6.15 ppm, quartet in the pro- markedly on the structures of the amino acid moieties of
ton-coupled spectrum) as the reaction intermediate. In ad- the N-phosphoryl amino acids. In the case of N-MMP-Val
dition, the reaction of N-MMP-Gly with acetic acid was (entry 8), the yield of dipeptide Val-Val is very low (1.0%),
analysed by ESI-MS and ESI-MS/MS (Figure 4). Acetyl whereas the yield of phosphate 4 is as high as 68.2%, as
phosphate 7 was observed at m/z = 198.9 as the [7 + Na]+ determined by 31P NMR integration. This finding can be
ion, which formed methyl phosphate (m/z = 156.9) and rationalized in terms of the steric effect of the hydrophobic
methyl metaphosphate (m/z = 124.9) by tandem MS/MS. side-chains of the amino acids that position the carboxylic
This assignment was further confirmed by comparison of acid and phosphoryl group closer and consequently facili-
3222
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3220–3228

Phosphoryl Transfer of N-Phosphoryl Amino Acids
Table 1. Yields of phosphates and peptides induced by N-phosphoryl amino acids.
Entry
Reactions[a] (m)
Yield of phosphate [%][b]
P2
Yield of peptide [%][c]
Pi
1
2
3
4
5
6
7
8
9
MMP-Gly (0.2)
5.1
5.0
3.0
5.7
4.1
4.4
40.4
68.2
5.7
5.2
6.6
5.2
6.3
11.8
17.2
5.7
8.3
3.6
G2 (13.0), G3 (1.2), G4 (0.1)
G2 (16.0), G3 (1.7), G4 (0.2)
G2 (11.5), G3 (1.0)
G2 (6.4), G3 (0.3)
G2 (6.8), G3 (0.3)
G2 (7.9), G3 (0.4)
A2 (4.6), A3 (0.2)
MMP-Gly (0.5)
MEP-Gly (0.2)[d]
MMP-Gly (0.2) + Mg2+ (0.02)
MMP-Gly (0.2) + Mg2+ (0.05)
MMP-Gly (0.2) + Mg2+ (0.1)
MMP-Ala (0.2)
MMP-Val (0.2)
MMP-Gly (0.2) + Ala (0.2)
V2 (1.0)
G2 (3.8), GA (4.0),[e]
AG (3.5), A2 (3.9)
[a] Reaction conditions: incubation at 40 °C, pD = 4.0–5.0, reaction time: entries 1, 3–6 and 9: 8 d; entry 7: 10 d; entries 2 and 8: 12 d.
For full experimental details, see the Supporting Information. [b] Yields were determined by integration of the 31P NMR spectra. Pi:
PO43–; P2: dimethyl pyrophosphate. [c] Peptide yields were determined relative to N-phosphoryl amino acids by HPLC. [d] MEP-Gly:
sodium salt of N-(ethoxyphosphoryl)glycine. [e] Including a small amount of triglycine.
formation of cyclic acylphosphoramidate (CAPA), which in
turn is hydrolysed to generate phosphate 4, rather than un-
favourable attack by another molecule of amino acid with
a large side-chain to generate an N-phosphoryl peptide.[10]
Significantly, the Group II ion Mg2+ could catalyse the for-
mation of dimethyl pyrophosphate 6 even at low concentra-
tion (5 mm). Yields of 6 were as high as 17.2% in the pres-
ence of 0.1 m Mg2+ with no formation of phosphate precipi-
tates (entry 6). Furthermore, it is noteworthy that free natu-
ral amino acids such as alanine can also be activated by N-
MMP-Gly to produce homo- and hetero-peptides in rea-
sonable yields (entry 9). These observations indicate marked
selectivity favouring the phosphoryl transfer reaction and
the notable dependence on the intrinsic structures of amino
acids for peptide formation.
A possible mechanism for this phosphoryl transfer reac-
tion is proposed in Scheme 2. Under slightly acidic condi-
tions, the phosphoryl group of an NPAA can undergo
spontaneous intramolecular transfer to carboxylic acid to
form pentacoordinate cyclic acylphosphoramidate interme-
diate 1a. This can be attacked not only by the nucleophilic
carboxylic group of acetic acid and amino acids at phos-
phorus to form “high-energy” anhydride phosphoester
bonds, but also by the amino group of any other amino
acid at the carbonyl group to produce N-phosphorylated
peptides, which subsequently hydrolyse to form oligopep-
tides. Promoted by the neighbouring carboxy group as an
essential component, the phosphoryl group of NPAAs may
be attacked nucleophilically to form pentacoordinate phos-
phorane intermediates through an addition/elimination
process (AN + DN in the IUPAC nomenclature), which con-
trasts with the unimolecular SN1-type mechanism (DN
+
AN) possibly involving a reactive monomeric metaphos-
phate intermediate.[23] Furthermore, it should be noted that
this phosphoryl transfer of N-phosphoryl amino acids with
the participation of the carboxylic acid group is different to
the general reactions of phosphoramidates by the concerted
mechanism (SN2) routes. Indeed, the active five-membered
Figure 3. Time-dependent, stacked 31P NMR spectra for the reac-
tions of N-MMP-Gly in D2O. (a) N-MMP-Gly (0.2 m) with acetic
acid (0.2 m); (b) N-MMP-Gly (0.2 m) with glycine (0.2 m).
tate nucleophilic attack of the carboxy on the phosphorus cyclic pentacoordinate phosphoric-amino acid mixed anhy-
to form pentacoordinate phosphate intermediate 1a drides have been observed by 31P NMR spectroscopy in
(Scheme 2). That leads to expulsion of a methanol with the anhydrous organic solvents.[6]
Eur. J. Org. Chem. 2011, 3220–3228
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3223

X. Gao, H. Deng, G. Tang, Y. Liu, P. Xu, Y. Zhao
FULL PAPER
Figure 4. ESI-MS spectra of the products from the reaction of N-MMP-Gly with acetic acid in D2O. (a) Mass spectrum acquired after
12 h; (b) ESI-MS/MS of [7 + Na]+ ion at m/z = 199.
(NFPA), o-phthaldialdehyde (OPA), 3-mercaptopropionic acid
(MPA) and trifluoroacetic acid (TFA) were purchased from Sigma
Conclusions
These studies suggest that N-phosphoryl amino acids are
a novel series of N-terminal-activated amino acids provid-
ing the energy source and phosphoryl donor not only for
intra- and intermolecular phosphoryl transfers to form
“high-energy” species (C-terminal-activated) such as pyro-
phosphates, acetyl phosphates and aminoacyl phosphates,
but also for the self-assembled synthesis of peptides under
mild aqueous conditions. Interestingly, unlike contempo-
rary protein biosynthetic pathways that need specific en-
zymes to catalyse the formation of aminoacyl adenylates,
NPAAs are capable of transferring their chemical energy
through the self-activation process to biologically signifi-
cant, energy-rich acyl phosphate species. Although the
yields of peptide formation and phosphoryl group transfer
are limited, the reactivities of N-phosphoryl amino acids as
small chemical models are novel. It is therefore possible that
the evolution of NPAAs may have been relevant to bio-
chemical energy metabolism and possibly to prebiotic
chemistry leading to the emergence of life. This study may
also contribute to a better understanding of the mechanism
of the intramolecular catalysis of biological phosphoryl
transfer reactions through possible intermediate penta-
covalent species and provide a chemical basis for the pharma-
cokinetics and metabolism of anti-viral and anti-cancer
amino acid phosphoramidate agents that may serve as leads
and scaffolds for medicinal research and development. Fur-
ther developments, such as the research of nucleotide N-
phosphoryl amino acids as novel chemical model systems
to elucidate the basic molecular recognition between amino
acids and nucleotides for the origin of the genetic code, are
currently under investigation in our group.
Chemical Co. (St. Louis, MO, USA). Diphenyl phosphite was ob-
tained from J&K Chemical Ltd. (Shanghai, China). HPLC-grade
methanol and acetonitrile were purchased from Tedia Company
Inc. (Fairfield, OH, USA). Strong acidic cation-exchange resin (Di-
aion SK1B) was purchased from H&E Co. Ltd. (Beijing, China).
Water was produced by the Milli-Q system (18 MΩ; Millipore,
Bedford, MA, USA). Unless specified otherwise, all chemicals and
solvents were analytical reagents.
1
NMR Analysis: H, 13C and 31P NMR spectra were recorded with
a Bruker 400 MHz NMR spectrometer. Chemical shifts for 1H
NMR are reported relative to internal tetramethylsilane (Me4Si, δ
= 0.00 ppm) with CDCl3 as solvent and D2O (H2O, δ = 4.70 ppm).
13C NMR spectra were recorded at 100 MHz. Chemical shifts for
13C NMR spectra were measured relative to CDCl3 (δ = 77.0 ppm).
31P NMR chemical shifts were recorded at 162 MHz, and chemical
shifts were relative to external 85% phosphoric acid (δ = 0.0 ppm).
Chemical shifts are given in ppm and signals are reported as s (sing-
let), d (doublet), t (triplet), q (quartet), quint. (quintuplet) and m
(multiplet).
ESI-MS and ESI-MS/MS Analysis: Mass spectra were acquired
with a Bruker Dalton Esquire 3000 plus ion-trap mass spectrometer
(Bruker Daltonik Co., Germany) equipped with a gas nebulizer
probe capable of analysing ions up to m/z = 6000. The mass spec-
trometer was operated in the positive ion mode. Nitrogen was used
as the dry gas at a flow rate of 4 L/min. The heated capillary tem-
perature was 250 °C. The reaction samples dissolved in methanol
were ionized by ESI and continuously infused into the ESI cham-
ber at a flow rate of 0.24 mL/h by a Cole-Parmer 74900 syringe
pump (Cole-Parmer Instrument Co., Vernon Hills, IL, USA). The
precursor ions [M + H]+ were selected by using an isolation width
of 1.0–1.5 m/z units and analysed by multistage tandem mass spec-
trometry (MSn) through collisions with helium.
High-resolution mass spectra were recorded with an ESI-Q-TOF-
MS spectrometer (Micromass, Waters, England) equipped with an
analytical electrospray source in the positive ion mode. The sample
in CH3OH was introduced into the source at a flow rate of 10 μL/
min. The nebulizing gas flow was 0.5 Lmin–1 and the ion accumu-
lation time was 10.0 ms.
Experimental Section
General: Commercial reagents were used as received. 15N-Glycine
was purchased from Cambridge Isotope Laboratories (Andover,
MA, USA). l-Amino acids were purchased from GL Biochem.
Ltd. (Shanghai, China). Gly-Gly (G2), Gly-Ala (GA), Ala-Gly
(AG), Ala-Ala (A2), Gly-Gly-Gly (G3), nonafluoropentanoic acid
LC-ESI-MS Analysis: To analyse the samples by LC-MS,[24] an
ion-trap mass spectrometer (Bruker Esquire 3000 plus, Dalton Co.,
Germany) equipped with an electrospray ionization source was
3224
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3220–3228

Phosphoryl Transfer of N-Phosphoryl Amino Acids
coupled to an Agilent 1100 binary pumping system (Agilent 1100
technologies, Wilmington, DE). Chromatogram column: Supelcosil
(Scheme 3).[26] Diphenyl phosphite (2 mmol) and
amount of Et3N (about five drops) were dissolved in dry THF
a catalytic
ABZ + plus 150ϫ4.6 mm I.D., 5 μm (Supelco, Bellefonte, PA, (15 mL) and cooled to –5 °C. CH3OH (1 mmol, 200 μL) in dry
USA). Solvent A: 2 mm NFPA in water; solvent B: acetonitrile. The
column was thermostatted at 25 °C and eluted by using an eluent
program with a flow rate of 1.0 mL/min at 4% B in 20 min. The
injection volume was 20 μL. Operating conditions for ESI in the
positive ion mode are listed below: spray voltage: 4000 V; target:
m/z = 200; capillary temperature: 280 °C; dry gas (N2): 10 L/min;
nebulizer (N2): 30 psi. Mass spectra were registered in the scan
range from m/z = 50 to m/z = 600. For LC-ESI-MS, about 1/10 of
the effluent from the UV absorbance detector was introduced into
the electrospray through a splitting T valve in order to avoid too
high a flow rate in the ion source.
THF (10 mL) was added dropwise to the solution under argon.
The resulting mixture was stirred at –5 °C for about 0.5 h and then
warmed to room temperature over 1 h. The progress of the reaction
was traced by 31P NMR spectroscopy. Then the amino acid methyl
ester hydrochloride (2 mmol), Et3N (3 mmol, 0.31 g) and C2Cl6
(3.6 mmol) in CH2Cl2 (15 mL) were sequentially added to the re-
sulting solution at 0 °C and the reaction mixture was stirred at
room temperature for about 4 h. The solvent was removed under
reduced pressure and the remaining residue was purified by silica
gel column chromatography (ethyl acetate/petroleum ether) to af-
ford the target products.
HPLC Analysis: The amino acids and peptides of the reaction mix-
ture were analysed by derivatization method with o-phthaldial-
dehyde (OPA)/3-mercaptopropionic acid (MPA) according to the
literature.[25] (a) The stock solution of OPA/MPA (prepared every
day): OPA (25 mg) + CH3OH (0.5 mL) + borate buffer (3.5 mL,
100 mmol/L, pH 10.4)
+ MPA (25 μL). (b) Borate buffer
(100 mmol/L, pH = 10.4). (c) Diluted reaction mixtures: the lyophi-
lized samples were dissolved in Milli-Q water (0.5 mL) and then
10 μL of the solution were diluted to 1.0 mL with Mili-Q water.
Derivatization procedure: 10 μL of (c) were added to solutions of
20 μL of (a) and 70 μL of (b). After 2 min, 25 μL aliquots were
injected into HPLC.
Scheme 3. Synthesis of the sodium salts of N-monoalkyloxyphos-
phoryl amino acids.
Reactions of Sodium Salts of N-(Alkoxyphosphoryl) Amino Acids
(N-MAP-AAs ) in Aqueous Solution
Methoxy Phenoxy Phosphoramidate of Glycine Methyl Ester (1a):
Colourless oil. H NMR (400 MHz, CDCl3): δ = 7.12–7.32 (m, 5
1
H), 3.81 (d, J = 17.2 Hz, 3 H), 3.73–3.78 (m, 2 H), 3.71 (s, 3
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 171.0 (d, J = 8 Hz),
150.7 (d, J = 6 Hz), 129.6, 124.8, 120.1 (d, J = 5 Hz), 53.6 (d, J =
5 Hz), 52.3, 42.8 ppm. 31P NMR (162 MHz, CDCl3): δ = 4.44 ppm.
(1) For Entries 1–3, 7 and 8: The sodium salts of N-MAP-AAs
(0.1 mmol; 0.25 mmol for entry 2) were dissolved in D2O (0.5 mL).
The pD of the solution was about 10.0–11.0 and then adjusted to
4.0–5.0 by the addition of 1 m DCl. The solution was kept at 40 °C
for several days and the pD of the solution was maintained at 4.0–
5.0 by frequent addition of 1 m DCl. The reaction was traced peri-
odically by 31P NMR spectrometry and ESI-MS through the reac-
tion process.
IR (film): ν = 3228, 2960, 2842, 1590, 1436, 1270, 1212, 1142,
˜
1038 cm–1. HRMS (ESI-TOF, positive ion mode): calcd. for [M +
H]+ 260.0699; found 260.0685; calcd. for [M + Na]+ 282.0507;
found 282.0508.
Methoxy Phenoxy [15N]Phosphoramidate of Glycine Methyl Ester
(15N-1a): Colourless oil. 1H NMR (400 MHz, CDCl3): δ = 7.11–
7.32 (m, 5 H), 3.80 (d, J = 11.6 Hz, 3 H), 3.70–3.77 (m, 2 H), 3.69
(s, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 170.0 (d, J =
8 Hz), 149.7 (d, J = 7 Hz), 128.6, 123.8, 119.1 (d, J = 5 Hz), 52.6
(d, J = 5 Hz), 51.3, 41.8 (d, J = 9 Hz) ppm. 31P NMR (162 MHz,
(2) For Entries 4–6: The sodium salt of N-MMP-Gly (0.1 mmol)
was dissolved in D2O (0.5 mL). MgCl2·6H2O (0.01–0.05 mmol) was
added to the solution. The pD of the solution was adjusted to 4.0–
5.0 by the addition of 1 m DCl. The solution was kept at 40 °C for
8 d. The reaction mixture was monitored by 31P NMR spec-
troscopy and analysed by LC-MS.
CDCl ): δ = 4.43 (d, J = 45.4 Hz) ppm. IR (film): ν = 3207, 2955,
˜
3
(3) For entry 9: The sodium salt of N-MMP-Gly (0.1 mmol) was
dissolved in D2O (0.5 mL). Alanine (0.1 mmol) was added to the
solution. The pD of the solution was adjusted to 4.0–5.0 by the
addition of 1 m DCl. The solution was kept at 40 °C for 8 d. The
reaction mixture was monitored by 31P NMR spectroscopy and
analysed by LC-MS.
1760, 1598, 1494, 1254, 1146, 1042 cm–1. HRMS (ESI-TOF, posi-
tive ion mode): calcd. for [M + H]+ 261.0658; found 261.0654.
Methoxy Phenoxy Phosphoramidate of Alanine Methyl Ester (10a):
The title compound was synthesized as a mixture of two dia-
1
stereomers. H NMR (400 MHz, CDCl3): δ = 7.12–7.28 (m, 5 H),
3.95–4.08 (m, 1 H), 3.81 (d, J = 11.6 Hz, 3 H), 3.71 (s, 3 H), 1.38
(d, J = 7.2 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 173.9
(d, J = 9 Hz), 150.7, 129.6 (d, J = 2 Hz), 124.8, 120.1 (d, J = 5 Hz),
53.6 (d, J = 6 Hz), 52.4, 50.1, 20.9 (d, J = 5 Hz) ppm. 31P NMR
All of the above reaction solutions were frozen, lyophilized and
stored at –20 °C for further HPLC analysis.
Reaction of Sodium Salt of N-(Methoxyphosphoryl)glycine and Ace-
tic Acid: The sodium salt of N-MMP-Gly (0.1 mmol) was dissolved
in D2O (0.5 mL). Acetic acid (0.1 mmol, about 7 μL) was added to
the solution. The solution was kept at 40 °C for 8 d. The reaction
was traced by 31P NMR spectroscopy and ESI-MS. The reaction
mixture was analysed by LC-MS.
(162 MHz, CDCl ): δ = 3.59, 3.49 ppm. IR (film): ν = 3211, 2977,
˜
3
1942, 1760, 1598, 1441, 1378, 1254, 1146, 1096, 1042, 922 cm–1.
HRMS (ESI-TOF, positive ion mode): calcd. for [M + H]+
274.0844; found 274.0791; calcd. for [M + Na]+ 296.0664; found
296.0657.
General Procedure for the Synthesis of the Alkoxy Phenoxy Phos-
phoramidates of Amino Acid Methyl Esters and the Alkoxy Phenoxy
Phosphoramidate of Glycylglycine Methyl Ester: The title com-
Methoxy Phenoxy Phosphoramidate of Valine Methyl Ester (11a):
The title compound was synthesized as a mixture of two dia-
stereomers. Colourless oil. 1H NMR (400 MHz, CDCl3): δ = 7.12–
7.33 (m, 5 H), 3.75–3.82 (m, 4 H), 3.70 (s, 3 H), 3.45–3.51 (m, 1 H),
pounds were synthesized according to
a literature method
Eur. J. Org. Chem. 2011, 3220–3228
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3225

X. Gao, H. Deng, G. Tang, Y. Liu, P. Xu, Y. Zhao
FULL PAPER
1.98–2.08 (m, 1 H), 0.87–0.94 (m, 6 H) ppm. 13C NMR (100 MHz,
(d, J = 10.8 Hz, 3 H), 1.28 (d, J = 6.8 Hz, 3 H) ppm. 13C NMR
CDCl3): δ = 173.1, 150.8, 129.5, 124.7, 120.7, 59.9 (d, J = 11 Hz), (100 MHz, D2O): δ = 182.9, 52.4, 51.7 (d, J = 8.0 Hz), 21.3 ppm.
53.7 (d, J = 6 Hz), 52.0.1, 32.19 (d, J = 4 Hz), 18.8, 17.3 (d, J =
31P NMR (162 MHz, D O): δ = 8.71 ppm. IR (KBr): ν = 3361,
˜
2
3 Hz) ppm. 31P NMR (162 MHz, CDCl3): δ = 4.44 ppm. IR (film): 2971, 1598, 1453, 1411, 1358, 1200, 1071 cm–1. HRMS (ESI-TOF,
ν = 3212, 2959, 1743, 1590, 1486, 1254, 1204, 1147, 1047, 926 cm–1.
positive ion mode): calcd. for [M + H]+ 228.0014; found 228.0017.
˜
HRMS (ESI-TOF, positive ion mode): calcd. for [M + H]+
302.1157; found 302.1152; calcd. for [M + Na]+ 324.0977; found
324.0964.
Sodium Salt of N-(Methoxyphosphoryl)valine (11): White solid; m.p.
1
Ͼ300 °C. H NMR (400 MHz, D2O): δ = 3.46 (d, J = 10.8 Hz, 3
H), 3.23 (dd, J = 9.6, 6 Hz, 1 H), 1.78–1.87 (m, 1 H), 0.91 (t, J =
6.8 Hz, 6 H) ppm. 13C NMR (100 MHz, D2O): δ = 182.0, 62.8,
51.6, 32.2 (d, J = 6.0 Hz), 18.8, 17.6 ppm. 31P NMR (162 MHz,
Ethoxy Phenoxy Phosphoramidate of Glycine Methyl Ester (12a):
1
Colourless oil. H NMR (400 MHz, CDCl3): δ = 7.13–7.24 (m, 5
D O): δ = 9.2 ppm. IR (KBr): ν = 3402, 2954, 1586, 1411, 1200,
˜
H), 4.16–4.23 (m, 2 H), 3.76–3.80 (m, 2 H), 3.71 (s, 3 H), 1.34 (dt,
J = 0.8, 6.8 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 173.9
(d, J = 9 Hz), 150.7, 129.6 (d, J = 2 Hz), 124.7, 120.1 (t, J = 5 Hz),
53.6 (d, J = 5 Hz), 52.4 (d, J = 2 Hz), 50.1 (d, J = 8 Hz), 20.9 (t,
J = 5 Hz) ppm. 31P NMR (162 MHz, CDCl3): δ = 2.94 ppm. IR
2
1076, 910 cm–1. HRMS (ESI-TOF, positive ion mode): calcd. for
[M + H]+ 256.0327; found 256.0330.
Sodium Salt of N-(Methoxyphosphoryl)glycine (12): White solid;
1
m.p. Ͼ300 °C. H NMR (400 MHz, D2O): δ = 3.78 (dq, J = 7.0,
(film): ν = 3228, 2950, 1756, 1590, 1486, 1212, 1146, 1047,
˜
8.4 Hz, 2 H), 3.33 (d, J = 8.0 Hz, 2 H), 1.16 (t, J = 7.2 Hz, 3
H) ppm. 13C NMR (100 MHz, D2O): δ = 179.2 (d, J = 10 Hz),
61.17, 45.09, 15.7 (d, J = 7 Hz) ppm. 31P NMR (162 MHz, D2O):
943 cm–1. HRMS (ESI-TOF, positive ion mode): calcd. for [M +
H]+ 274.0844; found 274.0840; calcd. for [M + Na]+ 296.0664;
found 296.0648.
δ = 8.41 ppm. IR (KBr): ν = 3299, 2967, 1586, 1399, 1308, 1254,
˜
1208, 1067 cm–1. HRMS (ESI-TOF, positive ion mode): calcd. for
[M + Na]+ 249.9833; found 292.9850.
Methoxy Phenoxy Phosphoramidate of Glycylglycine Methyl Ester
(2a): Colourless oil. 1H NMR (400 MHz, CDCl3): δ = 7.12–7.51
(m, 5 H), 4.70 (m, 1 H), 3.94 (dd, J = 5.6, 10.0 Hz, 2 H), 3.79 (d,
J = 11.2 Hz, 3 H), 3.66–3.71 (m, 5 H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 170.4 (d, J = 5.2 Hz), 170.1, 150.6 (d, J = 7 Hz), 129.7,
124.9, 120.1 (d, J = 4 Hz), 53.8 (d, J = 5 Hz), 52.1, 44.4, 40.9 ppm.
31P NMR (162 MHz, CDCl3): δ = 5.15 ppm. HRMS (ESI-TOF,
positive ion mode): calcd. for [M + Na]+ 339.0722; found 339.0724.
Sodium Salt of N-(Methoxyphosphoryl)glycylglycine (2): White so-
lid; m.p. 179–181 °C. 1H NMR (400 MHz, D2O): δ = 3.75 (s, 2 H),
3.49 (d, J = 10.4 Hz, 2 H), 3.47 (d, J = 10.8 Hz, 3 H) ppm. 13C
NMR (100 MHz, D2O): δ = 176.6, 174.6, 51.9, 44.5, 43.1 ppm. 31
P
NMR (162 MHz, D O): δ = 9.17 ppm. IR (KBr): ν = 3398, 2934
˜
2
1594, 1047, 1320, 1212, 1084, 1042 cm–1. HRMS (ESI-TOF, posi-
tive ion mode): calcd. for [M + H]+ 271.0072; found 271.0071;
calcd. for [M + Na]+ 292.9891; found 292.9883.
General Procedure for the Synthesis of the Sodium Salts of N-Alkyl-
oxyphosphoryl Amino Acids and N-(alkyloxyphosphoryl)glycyl-
glycine: The title compounds were synthesized according to a litera-
ture method.[9d] A solution of 0.4 m NaOH (CH3OH/H2O = 1:1 or
EtOH/H2O = 1:1, v/v, 10 mL) was added to the alkoxy phenoxy
phosphoramidate of the amino acid ester (or the alkoxy phenoxy
phosphoramidate of the glycylglycine methyl ester) (1 mmol). The
deprotection reactions were carried out at room temperature while
stirring under argon for 2–4 h. The course of the reaction was mon-
itored by 31P NMR until the disappearance of the starting material.
Once the starting material had disappeared, the solvent was re-
moved under reduced pressure to give an oily crude product, which
was recrystallized from 95% ethanol (about 10 mL). The precipi-
tate was washed three times with ethanol (3 mL) and dried under
vacuum to give the products as a white solid.
Disodium Salt of Methyl Phosphate (3): The title compound was
synthesized according to a literature method.[27] White solid; m.p.
1
81–82 °C. H NMR (400 MHz, D2O): δ = 3.53 (d, J = 10.8 Hz, 6
H) ppm. 13C NMR (100 MHz, D2O): δ = 52.8 (d, J = 5 Hz) ppm.
31P NMR (162 MHz, D2O): δ = 3.03 ppm.
Dimethyl Acetyl Phosphate: The title compound was synthesized
according to a reported procedure.[28] Acetyl chloride (1.95 g,
0.025 mol) and sodium dimethyl phosphate (3.7 g, 0.025 mol) were
suspended in dry tetrahydrofuran (25 mL). The resulting mixture
was stirred for 2 d at room temperature under nitrogen. The reac-
tion solution was filtered to remove sodium chloride. Removal of
1
the solvent left the target compound as a colourless oil. H NMR
(400 MHz, CDCl3): δ = 3.90 (d, J = 11.6 Hz, 6 H), 2.23 (d, J =
1.6 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 165.0 (d, J
= 9.2 Hz), 55.3 (d, J = 5 Hz), 21.7 (d, J = 8 Hz) ppm. 31P NMR
(162 MHz, CDCl3): δ = –6.2 ppm. MS (ESI, positive ion mode):
m/z = 168.9 [M + H]+, 190.9 [M + Na]+.
Sodium Salt of N-(Methoxyphosphoryl)glycine (1): White solid; m.p.
Ͼ300 °C. 1H NMR (400 MHz, D2O): δ = 3.39 (d, J = 8.4 Hz, 2
H), 3.51 (d, J = 10.8 Hz, 3 H) ppm. 13C NMR (100 MHz, D2O): δ
= 179.2, 51.8, 44.9 ppm. 31P NMR (162 MHz, D2O): δ = 9.64 ppm.
IR (KBr): ν = 3402, 2950, 1606, 1416, 1320, 1204, 1076 cm–1.
˜
Sodium Methyl Acetyl Phosphate (7): A solution of sodium iodide
(2.4 g, 16 mmol) in dry acetone (15 mL) was added to a solution
of acetyl dimethyl phosphate (2 g, 16 mmol) in dry acetone
(10 mL). The solution stood overnight at room temperature. The
precipitate was collected by filtration and washed with dry acetone
followed by CH2Cl2. Then the crude product was dried under vac-
uum to give the target product as a white powder. 1H NMR
(400 MHz, D2O): δ = 3.60 (d, J = 11.2 Hz, 3 H), 2.11 (d, J =
1.2 Hz, 3 H) ppm. 13C NMR (100 MHz, D2O): δ = 170.2 (d, J =
9.0 Hz), 53.6 (d, J = 6 Hz), 21.5 (d, J = 6 Hz) ppm. 31P NMR
(162 MHz, D2O): δ = –6.0 ppm. MS (ESI, positive ion mode): m/z
(%) = 176.9 [M + H]+. MS (ESI, negative ion mode): m/z = 152.8.
HRMS (ESI-TOF, positive ion mode): calcd. for [M + H]+
213.9857; found 213.9857; calcd. for [M + Na]+ 235.9677; found
235.9673.
Sodium Salt of [15N](Methoxyphosphoryl)glycine (15N-1): White so-
lid; m.p. Ͼ300 °C. 1H NMR (400 MHz, D2O): δ = 3.45 (d, J =
10.8 Hz, 3 H), 3.33 (d, J = 8.0 Hz, 2 H) ppm. 13C NMR (100 MHz,
D2O): δ = 179.2 (d, J = 9 Hz), 51.8, 45.0 (d, J = 7 Hz) ppm. 31P
NMR (162 MHz, D2O): δ = 9.67 (d, J = 32.4 Hz) ppm. 15N NMR
(40 MHz, D O): δ = 38.89 (d, J = 32.4 Hz) ppm. IR (KBr): ν =
˜
2
3253, 2942, 1602, 1411, 1370, 1308, 1125, 1067 cm–1. HRMS (ESI-
TOF, positive ion mode): calcd. for [M + H]+ 214.9828; found
214.9828.
Methyl Phosphate Dicyclohexylammonium Salt: The title com-
Sodium Salt of N-(Methoxyphosphoryl)alanine (10): White solid;
pound was synthesized according to a literature method.[29] Yield
1
1
m.p. Ͼ300 °C. H NMR (400 MHz, D2O): δ = 3.55 (m, 1 H), 3.49
55%. White solid; m.p. 203–205 °C. H NMR (400 MHz, D2O): δ
3226
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3220–3228

Phosphoryl Transfer of N-Phosphoryl Amino Acids
Mazza, J. Am. Chem. Soc. 1997, 119, 12089–12094; d) S. Tzvet-
kova, R. Kluger, J. Am. Chem. Soc. 2007, 129, 15848–15854.
a) P. V. Attwood, M. J. Piggott, X. L. Zu, P. G. Besant, Amino
Acids 2007, 32, 145–156; b) E. A. Ruben, M. S. Chapman, J. D.
Evanseck, J. Am. Chem. Soc. 2005, 127, 17789–17798; c) K.
Hakansson, D. B. Wigley, Proc. Natl. Acad. Sci. USA 1998, 95,
1505–1510.
a) Y. F. Zhao, P. S. Cao, J. Biol. Phys. 1994, 20, 283–287; b)
W. H. Zhou, Y. Ju, Y. F. Zhao, Q. G. Wang, G. A. Luo, Origins
Life Evol. Biosphere 1996, 26, 547–560; c) Z. Z. Chen, B. Tan,
Y. M. Li, Y. F. Zhao, J. Org. Chem. 2003, 68, 4052–4058.
H. Fu, Z. Z. Li, Y. F. Zhao, G. Z. Tu, J. Am. Chem. Soc. 1999,
121, 291–295.
= 3.44 (d, J = 10.4 Hz, 3 H), 3.08–3.15 (m, 2 H), 1.95–1.97 (m, 4
H), 1.75–1.78 (m, 4 H), 1.62 (d, J = 12.8 Hz, 2 H), 1.24–1.37 (m,
8 H), 1.10–1.19 (m, 2 H) ppm. 13C NMR (100 MHz, D2O): δ =
51.4 (d, J = 5 Hz), 50.2, 30.2, 24.2, 23.7 ppm. 31P NMR (162 MHz,
D2O): δ = 9.64 ppm.
[4]
[5]
Aminoacyl Methyl Phosphate: The title compound was synthesized
according to a literature method.[30] N,NЈ-Dicyclohexylcarbodiim-
ide (DCC, 0.43 g, 2.1 mmol) was added to a flask containing N-
Boc-amino acid (0.95 g, 4.4 mmol) in CH2Cl2 (30 mL) at room
temperature. The resulting reaction mixture was stirred for 5 min
under argon and methyl phosphate N,NЈ-diisopropyl-N-ethyl-
ammonium (DIEA) salt dissolved in CH3CN (5 mL) was added.
The reaction mixture was stirred at room temperature for 1 h. After
the reaction was complete, the resulting mixture was filtered and
washed with CH2Cl2 (10 mL). The CH2Cl2 solution was then ex-
tracted three times with water (18 mL). Then the aqueous phases
were combined, frozen and lyophilized. The product was purified
by short silica gel column chromatography using acetone as eluent
to remove inorganic salts. The solvent was removed in vacuo to
give N-Boc-amino acid methyl phosphate DIEA salt.
[6]
[7]
[8]
C. M. Cheng, X. H. Liu, Y. M. Li, Y. Ma, B. Tan, R. Wan,
Y. F. Zhao, Origins Life Evol. Biosphere 2004, 34, 455–464.
a) Y. Yamagata, H. Watanabe, M. Saitoh, T. Namba, Nature
1991, 352, 516–519; b) A. W. Schwartz, Philos. Trans. R. Soc.
London, Ser. B 2006, 361, 1743–1749; c) M. A. Pasek, T. P. Kee,
D. E. Bryant, A. A. Pavlov, J. I. Lunine, Angew. Chem. Int. Ed.
2008, 47, 7918–7920.
a) J. Rabinowitz, J. Flores, R. Krebsbach, G. Rogers, Nature
1969, 224, 795–796; b) X. Gao, Y. Liu, P. X. Xu, Y. M. Cai,
Y. F. Zhao, Amino Acids 2008, 34, 47–53; c) F. Ni, S. T. Sun,
C. Huang, Y. F. Zhao, Green Chem. 2009, 11, 569–573; d) F.
Ni, X. Gao, Z. X. Zhao, C. Huang, Y. F. Zhao, Eur. J. Org.
Chem. 2009, 18, 3026–3035.
a) S. J. Benkovic, P. A. Benkovic, J. Am. Chem. Soc. 1967, 89,
4714–4722; b) E. J. Sampson, J. Fedor, P. A. Benkovic, S.
Benkovic, J. Org. Chem. 1973, 38, 1301–1306.
F. Ni, W. Li, Y. M. Li, Y. F. Zhao, Curr. Protein Pept. Sci. 2005,
6, 437–442.
a) L. F. Landweber, Proc. Natl. Acad. Sci. USA 1999, 96,
11067–11068; b) R. Pascal, L. Boiteau, A. Commeyras, Top.
Curr. Chem. 2005, 259, 69–122.
a) C. R. Wagner, V. V. Iyer, E. J. McIntee, Med. Res. Rev. 2000,
20, 417–451; b) D. Cahard, C. McGuigan, J. Balzarini, Mini-
Rev. Med. Chem. 2004, 4, 371–381; c) S. J. Hecker, M. D. Erion,
J. Med. Chem. 2008, 51, 2328–2345; d) Y. Mehellou, J. Balzar-
ini, C. McGuigan, ChemMedChem 2009, 4, 1779–1791.
a) J. Balzarini, A. Karlsson, S. Aquaro, C. F. Perno, D. Cahard,
L. Naesens, E. D. Clercq, Proc. Natl. Acad. Sci. USA 1996, 93,
7295–7299; b) M. Derudas, D. Carta, A. Brancale, C. Vanpou-
ille, A. Lisco, L. Margolis, J. Balzarini, C. McGuigan, J. Med.
Chem. 2009, 52, 5520–5530; c) C. L. Chen, G. Yu, T. K. Venka-
tachalam, F. M. Uckun, Drug Metab. Dispos. 2002, 30, 1523–
1531.
a) O. Adelfinskaya, P. Herdewijn, Angew. Chem. Int. Ed. 2007,
46, 4356–4358; b) O. Adelfinskaya, M. Terrazas, M. Froeyen,
P. Marliere, K. Nauwelaerts, P. Herdewijn, Nucleic Acids Res.
2007, 35, 5060–5072; c) M. Terrazas, P. Marliere, P. Herdewijn,
Chem. Biodiversity 2008, 5, 31–39.
[9]
Aminoacyl methyl phosphate was obtained by adding a minimum
amount of TFA to the N-Boc-glycyl methyl phosphate. After
30 min, an excess of diethyl ether was added to precipitate the prod-
uct. Following centrifugation, the Et2O was decanted, the precipi-
tate was washed twice by agitation with Et2O followed by centrifu-
gation and decantation. The solid product was dried under vac-
uum.
[10]
[11]
[12]
1
Glycyl Methyl Phosphate (8): H NMR (400 MHz, D2O): δ = 3.90
(s, 2 H), 3.63 (d, J = 11.6 Hz, 3 H) ppm. 13C NMR (100 MHz,
D2O): δ = 164.7 (d, J = 9.0 Hz), 54.1 (d, J = 5 Hz), 40.7 (d, J =
8 Hz) ppm. 31P NMR (162 MHz, D2O): δ = –6.4 ppm. MS (ESI,
positive ion mode): m/z = 191.8 [M + H]+.
[13]
[14]
Alaninyl Methyl Phosphate (9): 1H NMR (400 MHz, D2O): δ =
4.19 (q, J = 7.6 Hz, 1 H), 3.61 (d, J = 11.6 Hz, 3 H), 1.54 (d, J =
7.2 Hz, 3 H) ppm. 13C NMR (100 MHz, D2O): δ = 164.7 (d, J =
9.0 Hz), 66.0, 54.1, 40.7 (d, J = 8.0 Hz) ppm. 31P NMR (162 MHz,
D2O): δ = –6.2 ppm. MS (ESI, positive ion mode): m/z = 205.9 [M
+ H]+.
Supporting Information (see footnote on the first page of this arti-
cle): LC-ESI-MS spectra of entry 1, 31P NMR spiking spectra of
products 2, 7, 8 and 9, HPLC chromatograms of entries 1–9 and
standard samples.
[15]
Acknowledgments
[16]
[17]
S. Michielssens, N. T. Trung, M. Froeyen, P. Herdewijn, M. T.
Nguyen, A. Ceulemans, Phys. Chem. Chem. Phys. 2009, 11,
7274–7285.
a) H. Fu, L. Xu, Q. Lv, J. Z. Wang, H. Z. Xiao, Y. F. Zhao,
Rapid Commun. Mass Spectrom. 2000, 14, 1813–1822; b) X.
Gao, F. Ni, J. Bao, Y. Liu, P. X. Xu, Y. F. Zhao, J. Mass Spec-
trom. 2010, 45, 779–787.
T. Lonnberg, M. Ora, H. Lonnberg, Mini-Rev. Org. Chem.
2010, 7, 33–43.
a) K. N. Allen, D. Dunaway-Mariano, Trends Biochem. Sci.
2004, 29, 495–503; b) P. P. Dzeja, R. Bortolon, C. Perez-Terzic,
E. L. Holmuhamedov, A. Terzic, Proc. Natl. Acad. Sci. USA
2002, 99, 10156–10161; c) J. R. Knowles, Annu. Rev. Biochem.
1980, 49, 877–919.
We are grateful for financial support from the National Natural
Science Foundation of China (NSFC) (grant numbers 21075103,
20972130, 20805037, and 20732004). We thank Prof. G. M. Black-
burn for helpful suggestions.
[1] a) A. R. Lewinsohn, M. Paecht-Horowitz, A. Katchalsky, Bi-
ochim. Biophys. Acta 1967, 140, 24–36; b) P. Schimmel, Annu.
Rev. Biochem. 1987, 56, 125–158.
[2] a) J. P. Biron, R. Pascal, J. Am. Chem. Soc. 2004, 126, 9198–
9199; b) J. P. Biron, A. L. Parkes, R. Pascal, J. D. Sutherland,
Angew. Chem. Int. Ed. 2005, 44, 6731–6734; c) L. J. Leman,
L. E. Orgel, M. R. Ghadiri, J. Am. Chem. Soc. 2006, 128, 20–
21.
[18]
[19]
[20]
[3] a) F. H. Westheimer, Science 1987, 235, 1173–1178; b)
N. S. M. D. Wickramasinghe, M. P. Staves, J. C. Lacey, Bio-
chemistry 1991, 30, 2768–2772; c) R. Kluger, R. W. Loo, V.
I. S. Kulaev, V. M. Vagabov, T. V. Kulakovskaya, The Biochem-
istry of Inorganic Polyphosphates, 2nd ed., Wiley, Chichester,
2004, pp. 194–204.
Eur. J. Org. Chem. 2011, 3220–3228
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3227

X. Gao, H. Deng, G. Tang, Y. Liu, P. Xu, Y. Zhao
FULL PAPER
[21] a) G. M. Blackburn, M. J. Brown, J. Am. Chem. Soc. 1969, 91,
525–526; b) S. A. Khan, A. J. Kirby, M. Wakselman, D. P.
Horning, J. M. Lawlor, J. Chem. Soc. B 1970, 1182–1187.
[22] F. Lipmann, Science 1954, 120, 855–865.
[23] a) A. C. Hengge, Acc. Chem. Res. 2002, 35, 105–112; b) M.
Ora, K. Mattila, T. Lonnberg, M. Oivanen, H. Lonnberg, J.
Am. Chem. Soc. 2002, 124, 14364–14372; c) M. J. P. Harger,
Chem. Commun. 2004, 1952–1953.
[24] K. Petritis, S. Brussaux, S. Guenu, C. Elfakir, M. Dreux, J.
Chromatogr. A 2002, 957, 173–185.
[25] a) B. N. Jones, S. Paabo, S. Stein, J. Liq. Chromatogr. 1981, 4,
565–586; b) A. Fiorino, G. Frigo, E. Cucchetti, J. Chromatogr.
1989, 476, 83–92.
[26]
[27]
[28]
X. S. Li, H. Fu, Y. Y. Jiang, Y. F. Zhao, J. Y. Liu, Synlett 2004,
14, 2600–2602.
P. K. Grzyska, P. G. Czyryca, J. Golightly, K. Small, P. Larsen,
R. H. Hoff, A. C. Hengge, J. Org. Chem. 2002, 67, 1214–1220.
R. Kluger, A. S. Grant, S. L. Bearne, M. R. Trachsel, J. Org.
Chem. 1990, 55, 2864–2868.
[29] C. Dueymes, C. Pirat, R. Pascal, Tetrahedron Lett. 2008, 49,
5300–5301.
[30] R. Kluger, L. L. Cameron, J. Am. Chem. Soc. 2002, 124, 3303–
3308.
Received: February 22, 2011
Published Online: May 2, 2011
3228
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3220–3228