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The Synthesis, Reactivity and NMR Investigation on 15N-Thiophosphoramidates (Supplementary Material)



Novel 15N-isotope enriched potassium and diammonium thiophosphoramidates were synthesized and their spectroscopic properties, along with reactivity towards several compounds, including histidine, thymidine, glucose and 2- deoxyribose are presented. The application of quantum mechanical DFT calculations for estimation of 31P NMR chemical shifts for several thiophosphoramidate ions and its derivatives are also discussed.
642 Letters in Organic Chemistry, 2009, 6, 642-647
1570-1786/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.
The Synthesis, Reactivity and NMR Investigation on 15N-Thiophospho-
Tomasz Ruman*,a, Karolina Dugopolskaa, Agata Jurkiewiczb, Dagmara Kramarza, Tomasz
Frczykc, Andrzej Leb,d and Wojciech Rodea,c
aRzeszów University of Technology, Faculty of Chemistry, Department of Biochemistry and Biotechnology, 6
Powstaców Warszawy Ave. 35-959 Rzeszów, Poland
bUniversity of Warsaw, Faculty of Chemistry, Quantum Chemistry Laboratory, 1 Pasteur St., 02-093 Warsaw, Poland
cNencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland
dPharmaceutical Research Institute, 8 Rydygier St., 01-793 Warsaw, Poland
Received February 28, 2009: Revised October 19, 2009: Accepted October 19, 2009
Abstract: Novel 15N-isotope enriched potassium and diammonium thiophosphoramidates were synthesized and their
spectroscopic properties, along with reactivity towards several compounds, including histidine, thymidine, glucose and 2-
deoxyribose are presented. The application of quantum mechanical DFT calculations for estimation of 31P NMR chemical
shifts for several thiophosphoramidate ions and its derivatives are also discussed.
Keywords: NMR, thiophosphate, tiophosphoramidate, thiophosphorylation.
Phosphates are ubiquitous in biochemistry, physico-
chemical properties allowing them to play a dominating role
in the living world [1]. Studies on phosphorylation/
dephosphorylation and biological activity of phosphorylated
compounds, as well as those aimed at drug development,
often take advantage of thiophosphate (or phosphorothioate)
analogues [2-5], offering, as compared to the phosphate
congeners, potential phosphorus-centered chirality and
increased stability towards chemical and enzymatic
hydrolysis. [6-8]. This hydrolytic stability is of particular
interest with studies on phosphorylation of proteins on basic
amino-acids, as due to their acid-labile character
phosphoramidates within proteins easily escape detection by
analytical methods [9, 10]. Moreover, phosphohistidine,
representing probably a major protein modification in
eukaryotes [9], applied as a hapten has proved too unstable
to generate antibodies [11]. Therefore methods are sought
allowing preparation of thiophos-phoramidate-modified
biomolecules under conditions mild enough to preserve
biological function [12, 13], in hope that thiophospho-
ramidates will be useful as more stable substitutes and
probes for the biological function of the corresponding
In the present study we synthesized 15N-thiophos-
phoramidates and tested their spectroscopic properties, as
well as potential to thiophosphorylate histidine, thymidine,
glucose and 2-deoxyribose.
*Address correspondence to the author at the Rzeszów University of
Technology, Faculty of Chemistry, Department of Biochemistry and
Biotechnology, 6 Powstaców Warszawy Ave. 35-959 Rzeszów, Poland,
The most common method of preparing O- and N-
thiophosphorylated compounds is the reaction of peptide
with a suitable kinase and -S-ATP [17]. A few non-
enzymatic approaches have been also described, namely the
on-resin phosphitylation-oxidation of a protected peptide and
the integration of a protected thiophosphorylated compound
by traditional Boc or Fmoc synthesis methods into a peptide
chain [18-20]. However, considering possible protein
thiophosphorylation, aimed at monitoring of the
modification’s influence on biological properties, the latter
methods do not allow reaction under mild enough
conditions. Another approach employed PSCl3 as a
thiophosphorylation agent [13] and allowed peptide
modification under mild conditions (room temperature, pH
8) but the hydrophobic character and wide range of reactivity
of thiophosphoryl chloride may cause the reaction sites to be
difficult to control. Moreover, PSCl3 and its hydrolysis
product PSCl2O- could react as a cross-linking or bridging
reagents. In contrast, the unifunctional reagents, anionic
thiophosphoramidates, in analogy to the parent
phosphoramidates [21], allow mild reaction conditions [12]
and should provide greater specificity.
The synthetic route applied to obtain 15N-enriched
potassium thiophosphoramidate (2, see Fig. (1)) was based
on that previously employed to synthesize the same
compound containing natural nitrogen isotope [14]. The first
reaction of the two-step synthesis is that of phosphorus
thiochloride with 15N-ammonium hydroxide solution
yielding 1 (
15N-diammonium thiophosphoramidate,
(15NH4)2PS(15NH2)O2). The 31P NMR spectrum of 1 in D2O
presents doublet resonance at 40.4 ppm belonging to
phosphorus from 1 and much smaller resonance of
hydrolysis product of 1 at 32.8 ppm. The resonance of 1 is in
a form of a doublet resulting from the heteronuclear coupling
The Synthesis, Reactivity and NMR Investigation Letters in Organic Chemistry, 2009, Vol. 6, No. 8 643
between 31P and 15N nuclei, each having nuclear spin of 1/2.
The analogical doublet resonance, corresponding to nitrogen
atom of amidate moiety was found in 15N NMR spectrum of
1 at 68.3 ppm. The one-bond 31P-15N coupling constant is
13.7 Hz being identical in both phosphorus and nitrogen
spectra. Besides, the same 15N NMR spectrum of 1 shows a
singlet resonance of ammonium ions at 19.9 ppm.
The second step of synthesis of 2, basically an ion
exchange reaction, is conducted at temperature of 60ºC. In
view of our results, this step could be potentially destructive
and was probably responsible for previously reported
unsuccessful synthesis of 2 [13]. The reaction with
potassium hydroxide, running under those conditions longer
than 5 min, leads to decomposition of thiophosphoramidate
due to hydrolysis of the P-N bond. The reaction time
exceeding 30min results in almost quantitative hydrolysis of
thiophosphoramidate to thiophosphate (1', Fig. (1)), the latter
showing its 31P NMR singlet resonance at 36.7 ppm. No
nitrogen resonances were found in 15N NMR spectrum of the
hydrolysis product, and this observation was confirmed by
other analytical methods demonstrating the absence of
nitrogen in the sample. With the reaction time of approx. 5
min, the second step of synthesis of 2 gave the expected
product with a good yield. The 31P NMR spectrum of 2 is
similar to that of 1 and presents a doublet resonance at 39.8
ppm, this chemical shift being in agreement with earlier
published data for the natural-nitrogen compound [14].
Thiophosphorylation of histidine using thiophos-
phoramidate (Fig. (2)) with natural nitrogen isotopes
abundance was presented by Pirrung et al. [12]. The
histidine derivatization presented in our work was conducted
with the use of 15N-enriched thiophosphoramidate that gives
more interesting mechanistic information. The mechanism of
this reaction contains an attack of the amino acid nitrogen
lone pair electrons on the phosphorus atom with elimination
of the amidate group that is 15N-enriched in our experiments.
The 31P NMR spectrum of the product shows a singlet
resonance of 3-thiophosphohistidine, confirming the
anticipated mechanism (vide supra). In accord with the
latter, no 15N resonances were found.
Potassium thiophosphoramidate was used for phos-
phorylation of saccharide hydroxyl groups of thymidine,
glucose and 2-deoxyribose. Thiophosphorylation of
thymidine leads to 5'-thiophosphate derivative (Fig. (2)) as
judged from NMR spectra. The 31P NMR spectrum of the
reaction mixture contains peaks (43.0 ppm), in the forms of a
doublet and asymmetrical singlet, which result from the
three-bond heteronuclear coupling between phosphorus and
two magnetically non-equivalent 5'-hydrogen atoms. The
explanation of this observation is the magnetic
nonequivalence of two hydrogen atoms from 5’-methylene
group. The nonequivalent hydrogen atoms couple to
thiophosphate phosphorus atom with different coupling
constant that results in doublet and asymmetrical singlet
resonances. The doublet form is a result of coupling of
phosphorus of spin with one hydrogen atom of spin.
Usually for saccharide methylene group one observes two
3JP-H couplings with different constants. The larger coupling
constant might result in clearly doublet form resonance,
while smaller coupling constant may give doublet or
unsymmetrical singlet resonance depending on resolution of
the spectrum.
The 3JP-H coupling constant for doublet resonance is in
the typical range for heteronuclear couplings of this type -
11.3 Hz [22]. The 31P NMR spectrum with hydrogen-
decoupling applied shows only one singlet resonance
pointing to the observed resonance pattern to originate from
a long-range P-H coupling. The latter observation was
confirmed by application of the gradient-enhanced 1H-31P
heteronuclear multiple bond correlation (HMBC) experiment
showing a strong coupling with 5'-hydrogen atoms at 3.82
ppm, in accord with previously published data [23].
Similar thiophosphorylation reaction was conducted for
glucose (Fig. (2)). The obtained 31P spectra present mainly
two doublets (40.0 and 39.4 ppm) with 3JP-H values of 10.3
Hz and 10.4 Hz for glucose-1-thiophosphate - and -
anomers, respectively. Similarly to thiophosphorylated
thymidine, the hydrogen decoupling reduces two doublets to
two singlets at averaged chemical shift positions proving that
the doublet resonance forms are due to proximity of 1'-
hydrogen atom. The gradient-enhanced 1H-31P HMBC
experiments allowed to find chemical shifts of hydrogen
atoms participating in coupling with phosphorus. The - and
-anomers of glucose-1-thiophosphate are in 1 to 9.5 molar
Cl Cl Cl P
OH 1. KOH / 60
2. AcOH
KOH / 1h, 60
Fig. (1). Synthesis of 1, 1' and 2.
644 Letters in Organic Chemistry, 2009, Vol. 6, No. 8 Ruman et al.
ratios respectively as calculated from integrals of resonances
[24, 25].
The 31P NMR spectrum of 2-deoxyribose thiophospho-
rylation product (Fig. (2)) also contains two doublets at 38.8
and 38.9 ppm with 3JP-H values of 11.3 and 10.4 Hz for 2-
deoxyribose-1-thiophosphate - and -anomers, respectively.
The - and -anomers of glucose-1-thiophosphate are in 1 to
2.5 molar ratios respectively. Similarly to glucose
thiophosphates, the gradient-enhanced 1H-31P HMBC and
hydrogen-decoupled 31P 1D experiment allowed the analysis
of the composition of the reaction mixture [25, 26].
In view of the foregoing, as well as previously published
data, potassium and ammonium thiophosphates are
derivation agents with a broad spectrum of use. Of particular
importance is fact that both compounds may be used under
conditions that should preserve protein structure. Recently
we found both potassium and diammonium thiophospho-
ramidates capable to thiophosphorylate His, Ser and Lys
residues (not shown) of Caenorhabditis elegans recombinant
thymidylate synthase protein [27].
Several model systems have been chosen for comparison
and for assessment of trends rather than attempting to
reproduce absolute values of NMR parameters. Such a
strategy stems from the fact that the calculation of shielding
constants and spin-spin coupling constants is extremely
sensitive to the choice of the molecular basis sets.
For most of the theoretical calculations the B3LYP/aug-
cc-pVTZ density functional theory was used. Due to the lack
of the standard aug-cc-pVTZ basis set for potassium, the
Sadlej's pVTZ basis set was used instead [28-32]. During the
course of geometry optimizations it was noted that the
mercaptophosphinyl tautomeric form of molecule 1'
(containing the P-SH bond) is less stable by 43.6 kJ mol-1
(10.4 kcal mol-1) than the phosphinothioyl form, containing
the formal P=S group. The potassium ion in this molecule is
located between the two nucleophilic centres: the O atom
and S atom (Supplementary Materials, Fig. (1A)). Another
interesting case was found for molecule 1 where the protons
originally attached to the NH4+ cations were finally
transferred to the oxygen atoms and the complex of
thiophosphoamide with two NH3 molecules arised
(Supplementary Materials, Fig. (2A)). The calculated
isotropic shielding constants are included in Table 1. The
degree of ionization of molecule 1 in the course of
hydrolysis is expected to be reflected in values of the 31P
isotropic shielding constants. For comparison, the 31P
isotropic shielding constants, calculated with the
B3LYP/aug-cc-pVTZ method, for phosphoric acid and its
ions are: 310.73 for H3PO4; 304.18 for H2PO4-, 286.57 for
HPO42- and 272.76 for PO43-. The isotropic shielding
2 15NH4+
Fig. (2). Thiophosphorylation products obtained by reaction with thiophosphoramidates. A = histidine, B = D-(+)-glucose, C = thymidine, D
= 2-deoxy-D-ribose.
The Synthesis, Reactivity and NMR Investigation Letters in Organic Chemistry, 2009, Vol. 6, No. 8 645
constants calculated for PSCl3 are 237.61 ppm (P) and
280.22 ppm (S). For molecule 2 the spin-spin coupling
constant was also calculated, giving 1JP-N=17.7 Hz.
As the accurate and reliable estimation of chemical shifts
for 31P NMR method is still long-term challenge, there is
very little knowledge describing thiophosphates and their
derivatives. The estimation of phosphorus chemical shift P
(Table 1, lowest line) based on comparison of electronic
shielding of particular atoms can be very useful information
regarding NMR properties of thiophosphates, a subject
poorly described in literature. The calculated 31P chemical
shifts for 1, 2 and 1’ in relation to H3PO4 (0.0 ppm) are
having similar values lying far above 35-45 ppm range
which is typical for thiophosphates. The only one value of
calculated chemical shift for 1-dianion seems to be in
thiophosphate range mentioned earlier. The similar
comparison of shieldings in relation to HPO42- dianion gives
much better results. The calculated chemical shifts for 1, 2
and 1’ (Q=0) in this case are 42.3, 40.9 and 39.5 ppm
respectively and are in much better agreement with
experimental results. Another important observation can be
made by comparison of shieldings of compounds with
formal phosphorus-sulfur double and single bonds. The
electronic shielding of ion 1’ has been calculated with both
formal double P=S bond with protonated oxygens
(phosphinothioyl tautomeric form, columns 6 and 7 in Table
1) and with single P-S bond (mercaptophosphinyl tautomeric
forms, columns 8 and 9 in Table 1). Observed and calculated
differences between those two forms are striking. The
calculated chemical shift of phosphinothioyl tautomer lays in
range typical for thiophosphates, but the P-SH tautomeric
form is upfield shifted and lies in 8-18 ppm range. This
observation can be related to very similar NMR properties of
S-substituted thiophosphate ions, for example with S-
thiophosphorylated sugars [25]. In conclusion, DFT methods
can be very useful anticipating NMR chemical shifts of
newly synthesized thiophosphate compounds as their 31P
NMR chemical shifts are found in quite large range starting
from +5 ppm, even to +80 ppm.
The importance of thiophosphate modification of
proteins and also smaller biologically active compounds has
been presented several times in literature. This paper
demonstrates that thiophosphoramidates are very promising
and useful derivation agents with vast range of possible
application. We have presented synthetic route to obtain
novel 15N-enriched potassium thiophosphoramidate and its
NMR properties with the use of both 31P- and 15N NMR
methods. The published synthetic route for potassium
thiophosphoramidate has been evaluated and optimized. It
was shown with the use of 15N NMR method that the
mechanism of thiophosphorylation of histidine and
presumably other amino acids involves an attack of the
amino acid nitrogen lone pair electrons on the phosphorus
atom with the elimination of amidate group. There are also
presented NMR properties of O-thiophosphorylation
products of thymidine, glucose and 2-deoxyribose. We also
present series of calculation results with the aim to estimate
the 31P NMR chemical shifts by analyzing electronic
shieldings of thiophosphoramidate and thiophosphate ions. It
has been shown that the chemical shift of thiophosphate ions
in phosphinothioyl tautomeric form lays in a range typical
for thiophosphates but the mercaptophosphinyl tautomer is
upfield shifted and lays in 8-18 ppm range, typical for S-
substituted thiophosphates.
All NMR spectra were obtained with Bruker Avance
spectrometer operating in the quadrature mode at 500.13
MHz for 1H, 202.46 MHz for 31P and 50.70 MHz for 15N
nuclei. The residual peaks of deuterated solvents were used
as internal standards in 1H NMR method. 31P and 15N NMR
spectra were recorded at 298K both with and without proton
decoupling. The internal standard used in 31P NMR was
inorganic phosphate (Pi) having its resonance at 2.14-2.16
ppm (at pH 7.8), 2.00-2.05 ppm (pH 7.5), 1.60-1.70 ppm (at
pH 5.0) and 0.0 ppm (at pH 1-1.5). All samples were
analyzed using gradient-enhanced 1H-31P Heteronuclear
Multiple Bond Correlation (HMBC) experiments. The
HMBC experiments were optimized for long range
Table 1. Isotropic Shielding Constants [ppm] Estimated with the B3LYP/aug-cc-pVTZ GIAO Calculations
1 2 1’
-OH, =S =O, -SH
Q = 0 Q=-1 Q = -2 Q = 0 Q = -1 Q = 0 Q = -1 Q = 0 Q = -1
P 244.3 258.0 266.7 245.8 247.0 247.1 245.7 297.9 295.3
S 648.3 617.7 643.6 624.4 641.7 649.0 654.2 553.6 559.6
O 170.7 168.0 126.6 130.5 146.8 137.0 149.1 111.0 108.3
O 163.1 147.5 126.6 184.4 173.2 169.8 145.4 118.1 133.8
O - - - - - 188.1 176.5 169.5 164.8
N 187.0 170.1 143.2 176.7 164.4 - - - -
P [ppm]
rel. to H3PO4 66.4 52.7 44.0 64.0 63.7 63.6 65.1 12.8 15.4
P [ppm]
rel. to HPO42- 42.3 28.5 19.9 40.8 39.5 39.5 40.9 11.4 8.7
The molecular total electric charge Q [a.u.] corresponds to the molecular complexes shown in the Scheme 1 (Q = 0) and to their respective anions (Q = -1 or -2) after removal of
1’ two
646 Letters in Organic Chemistry, 2009, Vol. 6, No. 8 Ruman et al.
couplings by using various 3JP-H values (1-20Hz). The 1H
NMR spectra were obtained with the use of HDO
suppression method. Elemental analysis was performed
using Elementar Vario EL-3 analyzer. FTIR spectra were
recorded on Perkin Elmer Paragon 1000 apparatus. The 15N-
ammonium hydroxide solution (15N>99%) was purchased
from SpectraGases Inc. Potassium thiophosphoramidate was
obtained using previously described method, slightly
modified with regard to the time of reaction (5-15 min) with
potassium hydroxide [14]. All other reagents and deuterated
solvents of the highest commercially available grade were
purchased from Aldrich and used without further
purification. Rubber septa joints were also purchased from
Aldrich. All procedures, including preparation of samples for
the NMR measurements, were carried out under nitrogen
atmosphere. The theoretical calculations have been
performed with the density functional B3LYP/aug-cc-pVTZ
method. The optimal geometries were obtained and
confirmed with positive harmonic frequencies. All the
calculations were performed with the Gaussian G03 (rev.
C.02) suite of programs [15]. The pictures of calculated
structures were made in the ChemCraft computer program
and are presented in Supplementary Materials [16].
15N-Thiophosphoramidate Diammonium Salt (1)
Thiophosphoryl chloride (0.25 ml, 2.45 mmol) was
added at 0ºC to 3N solution of 15NH4OH (4 ml, 12 mmol).
After 90 min. of vigorous stirring, the reaction mixture was
separated in a separatory funnel and to the aqueous layer
acetone (7 ml) added. Resulting mixture was stirred at 0ºC
for 60 min and filtered, to collect white precipitate that
formed. The product was then washed with two 10 ml
rations of tetrahydrofurane (cooled to 0ºC), then two 10 ml
rations of ethyl ether, and dried under high vacuum. Yield:
0.212 g (58%). Elemental analysis: H 6.68% (calculated
6.71%); N 29.92 (calculated 29.98%). 31P NMR (D2O,
[ppm]): 38.02 (1, d, 1J31P-15N = 13.7 Hz); 53.60 (PSCl3, s);
15N NMR (D2O, [ppm]): 68.34 (1, d, 1J15N-31P = 13.7 Hz);
19.92 (15NH4
+, s, LW 0.9 Hz).
15N-Potassium Thiophosphoramidate (2)
1 (0.2 g) was dissolved in 5 ml of 10% KOH and stirred
at 60ºC for 5 min. The solution was then cooled to room
temperature and brought to pH 6 with acetic acid. Ethanol
(20 ml) was added (at 0ºC), the solution stirred for 30 min,
and the resulting wh ite precipitate collected by filtration,
washed twice with tetrahydrofurane (10 ml rations) and dried
under high vacuum. Elemental analysis: H 1.97% (calculated
1.99%); N 9.84% (calculated 9.86%). 31P NMR (D2O,
[ppm]): 39.79 (d); FTIR (KBr, cm-1): 3391.8, 1656.3,
1571.9, 1410.2, 1091.0, 898.2, 871.8, 650.3, 623.3, 536.9.
Hydrolysis of 15N-Potassium Thiophosphoramidate
2 (50 mg) was dissolved in 0.6 ml of 50% KOH and
stirred at 60ºC for 60min. The solution was then cooled to
room temperature and brought to pH 6 with acetic acid.
Ethanol (20 ml) was added to obtained solution (at 0ºC), and
stirred for 30min. The white precipitate was collected by
filtration, washed with tetrahydrofurane (2 times 10 ml) and
dried under high vacuum. Potassium thiophosphate
(KH2PSO3, 1') was obtained as a main product. Elemental
analysis: H 1.40% (calculated 1.32%); N 0.0% (calculated
0.0%). 15N NMR (D2O, [ppm]): no 15N resonances found.
31P NMR (D2O, [ppm]): 36.70 (s).
15N-Thiophosphorylation of Histidine
To a sample of 10 mg histidine in 0.2M Tris-HCl buffer
(pH 7.5, 200 μL) potassium 15N-thiophosphoramidate was
added (2:1 potassium thiophosphoramidate to amino acid
molar ratio) and the reaction mixture shaken for 24 h at
298K. The NMR sample contained 200μL of reaction
solution and 400 μL of D2O. 31P NMR (D2O, [ppm]): 34.84
Thiophosphorylation of -D-Glucose
To a sample of 100 mg glucose in 300 μL of D2O
potassium thiophosphoramidate was added (3:1 potassium
thiophosphoramidate to saccharide molar ratio) and the
reaction mixture shaken for 24 h at 298K. The NMR sample
contained the reaction mixture and 300 μL of D2O. 31P NMR
(D2O, [ppm]): 39.35 (d, J=10.4Hz); 39.96 (d, J=10.3Hz).
Thiophosphorylation of Thymidine
To the sample containing 100 mg thymidine in 300 μL of
D2O, the potassium thiophosphoramidate was added (2.3:1
potassium thiophosphoramidate to saccharide molar ratio).
The sample was then shaken for 24 hours at 298K. The
NMR sample contained reaction mixture and 300 μL of D2O.
31P NMR (D2O, [ppm]): 43.00 (d+s, Jd=11.3Hz).
Thiophosphorylation of 2-Deoxyribose
To a sample of 100 mg 2-deoxyribose in 300 μL of D2O
potassium thiophosphoramidate was added (2.1:1 potassium
thiophosphoramidate to saccharide molar ratio) and the
resulting mixture shaken for 24 h at 298K. The NMR sample
contained the reaction mixture and 300 μL of D2O. 31P NMR
(D2O, [ppm]): 38.75 (d, J=11.3Hz); 38.93 (d, J=10.4Hz).
Supported by the Ministry of Education and Science,
Poland, Grant No. N204 088 31/2052 2006-2009. The ICM
computer center, University of Warsaw, Poland, is
acknowledged for the G18-6 computer grant.
Supplementary material is available on the publishers
Web site along with the published article.
[1] Westheimer, F.H. Science, 1987, 235, 1173.
[2] Eckstein, F. Annu. Rev. Biochem., 1985, 54, 367.
[3] Eckstein, F.; Gish, G. Trends Biochem. Sci., 1989, 14, 97.
[4] Heidenreich, O.; Pieken, W.; Eckstein, F. FASEB J., 1993, 7, 90.
The Synthesis, Reactivity and NMR Investigation Letters in Organic Chemistry, 2009, Vol. 6, No. 8 647
[5] Mescalchin, A.; Detzer, A.; Wecke, M.; Overhoff, M.; Wünsche,
W.; Sczakiel, G. Expert Opin. Biol. Ther., 2007, 7, 1531.
[6] Woniak, L.A.; Okruszek, A. Chem. Soc. Rev., 2003, 32, 158.
[7] Allen, J.J.; Lazerwith, S. E.; Shokat, K. M. J. Am. Chem. Soc.,
2005, 127(15), 5288.
[8] Zhao, Z. BBRC, 1996, 218, 480.
[9] Matthews, H.R. Pharmacol. Ther., 1995, 67, 323.
[10] Klumpp, S.; Krieglstein, J. Biochim. Biophys. Acta, 2005, 17, 291.
[11] Hunter, T. in Life Sciences for the 21st Century; E. Keinan, I.
Schechter, M. Sela (eds.), Wiley-VCH; Weinheim, 2004; pp. 191-
[12] Pirrung, M.C.; James, K.D.; Rana, V.S. J. Org. Chem., 2000, 65,
[13] Lasker, M.; Bui, C.D.; Besant, P.G.; Sugawara, K.; Thai, P.;
Medzihradszky, G.; Turck, C.W. Protein Sci., 1999, 8(10), 2177.
[14] Katagi, T. J. Mol. Struct.: Theochem, 1990, 68, 61.
[15] Gaussian 03, Revision C.02; Frisch, M. J.; Trucks, G. W.; Schlegel,
H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.;
Montgomery, Jr. J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.;
Millam, J. M.; Iyengar, S.S.; Tomasi, J. ; Barone, V.; Mennucci,
B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji,
H.; Hada, M.; Ehara, M. ; Toyota, K.; Fukuda, R. ; Hasegawa, J.;
Ishida, M. Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene,
M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C. ;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin,
A.J.; Cammi, R.; Pomelli, C.; Ochterski, J.W.; Ayala, P.Y.;
Morokuma, K.; Voth, G.A.; Salvador, P.; Dannenberg, J.J.;
Zakrzewski, V.G.; Dapprich, S.; Daniels, A.D.; Strain, M.C.;
Farkas, O.; Malick, D.K.; Rabuck, A.D.; Raghavachari, K.;
Foresman, J.B.; Ortiz, J.V.; Cui, Q.; Baboul, A.G.; Clifford, S.;
Cioslowski, J.; Stefanov, B.B.; Liu, G.; Liashenko, A.; Piskorz, P.;
Komaromi, I.; Martin, R.L.; Fox, D.J.; Keith, T.; Al-Laham, M.A.;
Peng, C.Y.; Nanayakkara, A.; Challacombe, M.; Gill, P.M.W.;
Johnson, B.; Chen, W.; Wong, M.W.; Gonzalez, C.; Pople, J.A.,
Gaussian, Inc., Wallingford CT, 2004.
[16] (accessed January 5th 2009, 9:00).
[17] Frey, P. A. Adv. Enzymol., 1989, 62, 119.
[18] Mora, N.; Lacombe, J.M.; Pavia, A.A. Int. J. Pept. Prot. Res.,
1995, 45, 53.
[19] Aemissegger, A.; Carrigan, C.N.; Imperiali, B. Tetrahedron, 2007,
63, 6185.
[20] Guzacev, A. P.; Manoharan, M. Tetrah. Lett., 2001, 42, 4769.
[21] Medzihradszky, K.F.; Phillips, N.J.; Senderowicz, L.; Wang, P.;
Turck, C.W. Protein Sci., 1997, 6, 1405.
[22] Isab, A.A.; Hussain, M.S.; Akhtar, M.N.; Wazeer, M.I.M.; Al-
Arfaj, A.R. Polyhedron, 1999, 18, 1401.
[23] Jankowska, J.; Sobkowska, A.; Cielak, J.; Sobkowski, M.;
Kraszewski, A.; Skawiski, J.; Shugar, D. J. Org. Chem., 1998, 63,
[24] Singh A.N.; Newborn, J.S.; Rauschel, F.M. Bioorg. Chem., 1988,
16, 206.
[25] Ratcliffe, R.G.; Sachar-Hill, Y. Biol. Rev., 2005, 80, 27.
[26] Knight, W. B.; Sem, D.S.; Smith, K.; Miziorko, H.M.; Rendina, A.
R.; Cleland, W.W. Biochemistry, 1991, 30, 4970.
[27] Wiska, P.; Goos, B.; Ciela, J.; Zieliski, Z.; Frczyk, T.;
Waajtys-Rode, E.; Rode, W. Parasitology, 2005, 131, 247.
[28] (accessed January 3rd, 2009, 15:00).
[29] Sadlej, A.J. Collec. Czech. Chem. Commun., 1988, 53, 1995.
[30] Sadlej, A.J.; Urban, M. J. Mol. Struct.: Theochem, 1991, 234, 147.
[31] Sadlej, A.J. Theor. Chim. Acta, 1992, 81, 45.
[32] Sadlej, A.J. Theor. Chim. Acta, 1992, 81, 339.
... Hz) is carbon from NH-N-C. The resonance of phosphorous is in a form of two multiplets resulting from the heteronuclear coupling between 31 P and 15 N nuclei and then 31 P and 1 H and 31 P and 13 C. [26] In Fig. 2, the FTIR data of CN-3 (top) show the absorption of NH at 3169 cm À1 , which is very much the same position as in normal amines. The NH stretching and deformation vibrations are barely affected by the presence of the phosphorous atom. ...
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A novel flame retardant diethyl 4-methylpiperazin-1-ylphosphoramidate (CN-3) containing phosphorous and nitrogen was prepared. Its chemical structure was confirmed by nuclear magnetic resonance (1H-, 13C-, and 31P-NMR), Fourier transform infrared spectroscopy, and elemental analysis. Print cloth and twill fabrics were treated with CN-3 to achieve different levels of add-on (722?wt% add-ons for print cloth and 318?wt% add-ons for twill). Thermogravimetric analysis, vertical flame test, and limiting oxygen index (LOI) were performed on the treated cotton fabrics and showed promising results. When the treated print cloth and twill fabric samples were tested using the vertical flame test (ASTM D6413-08), we observed that the ignited fabrics self-extinguished and left behind a streak of char. Treated higher add-ons fabrics were neither consumed by flame nor produced glowing ambers upon self-extinguishing. LOI (ASTM 286309) was used to determine the effectiveness of the flame retardant on the treated fabrics. LOI values increased from 18?vol% oxygen in nitrogen for untreated print cloth and twill fabrics to maximum of 28 and 31?wt% for the highest add-ons of print cloth and twill, respectively. The results from cotton fabrics treated with CN-3 demonstrated a higher LOI value as well as a higher char yield because of the effectiveness of phosphorus and nitrogen as a flame retardant for cotton fabrics. Furthermore, FT-IR and SEM were used to characterize the chemical structure on the treated fabrics as well as the surface morphology of char areas of treated and untreated fabrics. Published 2012. This article is a US Government work and is in the public domain in the USA.
This is the first part of two closely related reviews dealing with the computation of phosphorus-31 nuclear magnetic resonance chemical shifts in a wide series of organophosphorus compounds including complexes, clusters, and bioorganic phosphorus compounds. In particular, the analysis of the accuracy factors, such as substitution effects, solvent effects, vibrational corrections, and relativistic effects, is presented. This review is dedicated to the Full Member of the Russian Academy of Sciences Professor Boris A. Trofimov in view of his invaluable contribution to the field of synthesis, nuclear magnetic resonance, and computation studies of organophosphorus compounds.
Protein phosphorylation is a ubiquitous posttranslational modification that regulates cell signaling in both prokaryotes and eukaryotes. Although the study of phosphorylation has made great progress, several major hurdles remain, including the difficulty of the assignment of endogenous substrates to a discrete kinase and of global phosphoproteomics investigations. We have developed a novel chemical strategy for detecting phosphorylated proteins. This method utilizes adenosine 5'-O-(3-thiotriphosphate) (ATPγS), which results in the transfer of a thiophosphate moiety by a kinase to its substrate(s). This group can subsequently be employed as a nucleophilic handle to promote protein detection. To selectively label thiophosphorylated proteins, cellular thiols (e.g. cysteine-containing proteins) must first be blocked. Most common cysteine-capping strategies rely upon the nucleophilicity of the sulfur group and would therefore also modify the thiophosphate moiety. We hypothesized that the radical-mediated thiol-ene reaction, however, would be selective for cysteine over thiophosphorylated amino acids due to the differences in the electronics and pKa values between these groups. Here, we report rapid and specific tagging of thiophosphorylated proteins in vitro following chemoselective thiol capping using the thiol-ene reaction.
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Reversible phosphorylation is the most widespread posttranslational protein modification, playing regulatory role in almost every aspect of cell life. The majority of protein phosphorylation research has been focused on serine, threonine and tyrosine that form acid-stable phosphomonoesters. However, protein histidine, arginine and lysine residues also may undergo phosphorylation to yield acid-labile phosphoramidates, most often remaining undetected in conventional studies of protein phosphorylation. It has become increasingly evident that acid-labile protein phosphorylations play important roles in signal transduction and other regulatory processes. Beside acting as high-energy intermediates in the transfer of the phosphoryl group from donor to acceptor molecules, phosphohistidines have been found so far in histone H4, heterotrimeric G proteins, ion channel KCa3.1, annexin 1, P-selectin and myelin basic protein, as well as in recombinant thymidylate synthase expressed in bacterial cells. Phosphoarginines occur in histone H3, myelin basic protein and capsidic protein VP12 of granulosis virus, whereas phospholysine in histone H1. This overview of the current knowledge on phosphorylation of protein basic amino-acid residues takes into consideration its proved or possible roles in cell functioning. Specific requirements of studies on acid-labile protein phosphorylation are also indicated.
In search of an activity-preserving protein thiophosphorylation method, with thymidylate synthase recombinant protein used as a substrate, potassium thiophosphoramidate and diammonium thiophosphoramidate salts in Tris- and ammonium carbonate based buffer solutions were employed, proving to serve as a non-destructive environment. Using potassium phosphoramidate or diammonium thiophosphoramidate, a series of phosphorylated and thiophosphorylated amino acid derivatives was prepared, helping, together with computational (using density functional theory, DFT) estimation of (31)P NMR chemical shifts, to assign thiophosphorylated protein NMR resonances and prove the presence of thiophosphorylated lysine, serine and histidine moieties. Methods useful for prediction of (31)P NMR chemical shifts of thiophosphorylated amino acid moieties, and thiophosphates in general, are also presented. The preliminary results obtained from trypsin digestion of enzyme shows peak at m/z 1825.805 which is in perfect agreement with the simulated isotopic pattern distributions for monothiophosphate of TVQQQVHLNQDEYK where thiophosphate moiety is attached to histidine (His(26)) or lysine (Lys(33)) side-chain.
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This manuscript summarizes the results of studies on the application of the reaction of dialkyl (aryl) phosphor-amidate anions with carbonyl electrophiles for stereospe-cific synthesis of P-chiral biophosphates (Stec reaction). Following the results obtained with organic phosphor-amidates which delineated the scope of the reaction and its stereochemical course, the application of the title reaction is presented for the preparation of diastereomerically pure P-chiral cyclic nucleotide analogues (phosphorothioates, phos-phoroselenoates, phosphoroselenothioates, isotopomeric 18 O-phosphates), and P-chiral nucleoside monophosphate analogues, as well as dinucleoside phosphate analogues (phosphorothioates, methanephosphonates).
Methods have been developed for the enzymatic synthesis of α-glucose 1-thiophosphate, α-galactose 1-thiophosphate, glucose 6-thiophosphate, uridine-5′-O-(2-thiodiphosphoglucose), and uridine-5′-O-(2-thiodiphosphogalactose). The purified compounds were shown to be substrates for sucrose phosphorylase, glactokinase, UDP-glucose pyrophosphorylase, galactose-1-phosphate uridyltransferase, hexokinase, glucose-6-phosphate dehydrogenase, UDP-glucose dehydrogenase, and UDP-galactose-4-epimerase. The formation of uridine-5′-O-(2-thiodiphosphoglucose) from α-glucose 1-thiophosphate and UTP as catalyzed by UDP-glucose pyrophosphorylase produces only one of the two possible epimers at the β-thiophosphoryl position. The absolute stereochemistry has not been determined. Uridine-5′-O-(2-thiodiphosphoglucose) was not utilized as an alternate substrate in the transfer of a glucosyl group in the reactions catalyzed by glycogen synthetase and sucrose synthetase. The incubation of cyanate and thiophosphate at pH 5.0 was found to result in the rapid removal of sulfur from the thiphosphate. The reaction is stoichiometric with respect to cyanate and thiophosphate. The data are consistent with the rapid formation and hydrolysis of thiocarbamoyl phosphate.
The disproportionation of cyanogold(I) complexes of general formula [R3PAu13C15N] forming [(R3P)2Au]+ and [Au(13C15N)2]− ions has been investigated using 13C, 15N and 31P NMR spectroscopy for a series of phosphines with R=cyclohexyl, i-propyl, Et, Me, cyclohexyl/diphenyl, o-tolyl, p-tolyl, m-tolyl, p-tolyl/diphenyl, allyl/diphenyl, phenyl, tri(cyanoethyl) CEP, and 1-naphthyl. The 13C NMR of the 13C15N group in these complexes exhibited two distinct resonances, one due 13C in the starting [R3PAu13C15N] complex and the second in the [Au(13C15N)2]− anion. The 31P NMR spectra revealed two 31P resonances due to [R3PAu13C15N] complex, and the [(R3P)2Au]+ cation. The 15N NMR revealed only an averaged resonance due to [R3PAu13C15N] and [Au(13C15N)2]− anion, except in the cases of [Me3PAu13C15N] and [Et3PAu13C15N] where two resonances were observed. The coupling constants, 1J(13C–15N), 2J(31P–13C) and 3J(31P–15N) were obtained for all complexes and the free energies of activation for ligand disproportionation were determined using 31P–{1H} NMR band shape analysis.
A simple and efficient protocol for the preparation of unprotected nucleoside 5‘-H-phosphonates and nucleoside 5‘-H-phosphonothioates via a one-step deprotection of suitable precursors with methylamine has been developed. The synthetic utility of the unprotected nucleotide derivatives was demonstrated by converting them under mild conditions to the corresponding nucleoside 5‘-phosphorothioate and nucleoside 5‘-phosphorodithioate monoesters. Factors affecting oxidation of H-phosphonate, H-phosphonothioate, and phosphite derivatives with elemental sulfur are also discussed.
Two novel phosphoramidite building blocks and a solid support that allow an efficient solid-phase phosphorylation or thiophosphorylation of synthetic oligonucleotides were developed. The utility of these synthetic tools was demonstrated in the preparation of 5%-or 3%-thiophosphorylated oligonucleotides, which were subsequently labeled at the termini with fluorescent reporters. Terminal thiophosphate (PS) group provides a conve-nient site for regiospecific conjugation of synthetic oligonucleotides to a variety of ligands bearing an electrophilic group. 1,2 However, extensive application of this technique has been hampered by the lack of efficient methods for the terminal thiophosphorylation of synthetic oligonucleotides. Although convenient methods for the 5%-3–5 and 3%-phosphorylation 5,6 are well documented in the literature, those dealing with the introduction of the terminal PS group are limited. The release of the terminal thionophosphomonoester employs either reductive 7 or oxidative 1 conditions that unnecessarily complicate the deprotection of oligonu-cleotides. Besides, both approaches suffer from substan-tial desulfurization of the PS moiety, which results in the corresponding oligonucleotide 5%-or 3%-phosphate (PO) in ca. 15% yield. 1 The subsequent removal of this side product by HPLC purification is not trivial. We report here a convenient approach for the terminal phosphorylation and thiophosphorylation of synthetic oligonucleotides using phosphoramidite building blocks 1a and 1b and a solid support 2. In the case of thiophosphorylation, the final deprotection under stan-dard conditions results in the desired products with only minor desulfurization (1–1.5%). The synthesized oligonucleotides were successfully converted to their fluorescent conjugates by reacting with iodoacetamide derivatives of fluorescent dyes, fluorescein and pyrene.
The synthesis of 1-(2-nitrophenylethyl) caged O-phosphorothioylserine, -threonine and -tyrosine derivatives is reported. These amino acid building blocks can be directly incorporated into peptides by Fmoc-based solid phase synthesis as their pentafluorophenyl esters or as symmetric anhydrides. Upon irradiation with UV light, the thiophosphate group, representing a hydrolysis resistant phosphate analog, is revealed.
A number of phosphorylated thiosugars have been prepared and tested as substrates for metabolic reactions. 6-Thioglucose-6-P is readily synthesized by reaction of 6-tosylglucose with trisodium thiophosphate at pH 10 in aqueous solution; the product has only sulfur between carbon and phosphorus. When ethyl glycerate is tosylated and treated similarly with thiophosphate, a 5:1 mixture of 3-thioglycerate-3-P and the 2-isomer is formed. 6-Thioglucose-6-P is converted by glycolytic enzymes to triose phosphates, 3-thioglycerol-3-P and 3-thioglycerate-3-P, and is oxidized by enzymes of the hexose monophosphate shunt to 5-thioribulose-5-P, which can be converted via phosphoribulokinase and ribulose-bis-P carboxylase into 3-P-glycerate and 3-thioglycerate-3-P. For most of the non-phosphoryl-transferring enzymes there are only moderate effects on Vmax and Km. Phosphoglucoisomerase, however, is very sensitive to the sulfur for oxygen change, with Vmax decreasing 60-fold and Km increasing 15-fold. Surprisingly, phosphoribulokinase has a V/K value for 5-thioribulose-5-P that is over 3 orders of magnitude less than for ribulose-5-P. 6-Thio-glucose-6-P was found to be a substrate for several enzymes that transfer the phosphoryl group. It is as good a substrate for alkaline phosphatase as glucose-6-P, and with phosphoglucomutase it is converted to 6-thioglucose-1-P with a rate that is 11% of the rate of reaction of glucose-1-P, with a Keq value of 45.6. The free energy of hydrolysis of the phosphorylated thiol is thus -7.2 kcal/mol at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)
Several phosphoserine, phosphothreonine and phosphotyrosine synthons suitable for the stepwise synthesis of phosphopeptides were prepared. Treatment of methylthiomethyl (MTM) esters of either Z-, Boc-, Allocserine and threonine with phosphochloridate in pyridine followed by MgBr2 cleavage of MTM in diethyl ether afforded the title compounds in good yield. Thiophosphoserine and phosphotyrosine synthons were also obtained by the phosphoramidite method using di-(2,2,2-trichloroethyl)-N,N-diisopropylphosphoramidite and MCPBA as oxidizing reagent. Trichloroethyl proved valuable as phosphate protecting group especially in phosphotyrosine derivatives owing to its stability in acidic conditions. These synthons were involved in the liquid-phase synthesis of several phospho and/or thiophosphopeptides related to either src-protein kinase or rat liver pyruvate kinase.
The modification of phosphate into phosphorothioate internucleotidic linkages in various RNAs and their usefulness in identifying phosphate positions essential for function are described. Several modifications of the 2'-hydroxyl group of the ribose, particularly the replacement by fluorine atoms and amino groups, is discussed. These studies have been concentrated on hammerhead ribozymes in order to determine hydroxyl groups important for the catalytic activity. In addition these derivatives have been instrumental in rendering ribozymes more stable toward nucleases.