Thiophosphorylation of free amino acids and enzyme protein by thiophosphoramidate ions

Article abstract of DOI:10.1016/j.bioorg.2009.11.002

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 ³¹P 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 ³¹P 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 (His²⁶) or lysine (Lys³³) side-chain.

Full text of DOI:10.1016/j.bioorg.2009.11.002

Bioorganic Chemistry 38 (2010) 74–80
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Thiophosphorylation of free amino acids and enzyme protein
by thiophosphoramidate ions
a
b
a
c
Tomasz Ruman a, , Karolina Długopolska , Agata Jurkiewicz , Dagmara Rut , Tomasz Fra˛czyk ,
*
Joanna Ciesla , Andrzej Les b,d, Zbigniew Szewczuk e, Wojciech Rode a,c
c
´
´
a
´
Rzeszów University of Technology, Faculty of Chemistry, Department of Biochemistry and Biotechnology, 6 Powstanców Warszawy Ave., 35-959 Rzeszów, Poland
b University of Warsaw, Faculty of Chemistry, Quantum Chemistry Laboratory, 1 Pasteur Street, 02-093 Warsaw, Poland
c Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland
d Pharmaceutical Research Institute, 8 Rydygier St., 01-793 Warsaw, Poland
e University of Wroclaw, Faculty of Chemistry, ul. F. Joliot-Curie 14, 50-383 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 October 2009
Available online 26 November 2009
In search of an activity-preserving protein thiophosphorylation method, with thymidylate synthase
recombinant protein used as a substrate, potassium thiophosphoramidate and diammonium thiophosph-
oramidate salts in Tris- and ammonium carbonate based buffer solutions were employed, proving to
serve as a non-destructive environment. Using potassium phosphoramidate or diammonium thiophosph-
oramidate, a series of phosphorylated and thiophosphorylated amino acid derivatives was prepared,
helping, together with computational (using density functional theory, DFT) estimation of 31P 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 31P NMR chem-
ical 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 (His26) or lysine (Lys33) side-
chain.
Keywords:
NMR
Thiophosphate
Tiophosphoramidate
Thiophosphorylation
Thymidylate synthase
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
biomolecules under conditions mild enough to preserve biological
function [12,13].
Physicochemical properties of phosphates allow them to play a
dominating role in the living world [1]. Thiophosphate (or phosp-
horothioate) analogues, offering, as compared to the phosphate
congeners, potential phosphorus-centered chirality and increased
stability towards chemical and enzymatic hydrolysis [2–4], are
often used in studies on biological activity of phosphorylated com-
pounds [5–8]. The hydrolytic stability is of particular interest with
studies on phosphorylation of protein basic amino acids, as due to
their acid-labile character phosphoramidates within proteins eas-
ily escape detection by analytical methods [9,10]. Moreover, phos-
phohistidine, representing probably a major protein modification
in eukaryotes [9], applied as a hapten has proved too unstable to
generate antibodies [11]. In hope that thiophosphoramidates will
be useful as more stable substitutes and probes for the biological
function of the corresponding phosphoramidates, methods are
sought allowing preparation of thiophosphoramidate-modified
The first thiophosphorylation of peptide was described by Las-
ker et al. [12]. Surprisingly, despite the successful preparation of
thiophosphohistidine by reaction with thiophosphoramidate, the
authors were unable to thiophosphorylate histidine-rich peptide.
Therefore they developed another method, employing PSCl3 as a
phosphorylation reagent.
The present study was done in search of an activity-preserving
protein thiophosphorylation, with thymidylate synthase recombi-
nant protein, found to undergo acid-labile phosphorylation [14],
used as a substrate. Instead of phosphorus thiochloride that (i)
shows high reactivity towards popular buffer ingredients, (ii) pro-
duces acidic conditions when used in water solutions and (iii)
could act as a cross-linking agent giving products difficult to
analyze [12], 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 (DFT) estimation of 31P
* Corresponding author. Fax: +48 178543655.
E-mail address: tomruman@prz.edu.pl (T. Ruman).
0045-2068/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2009.11.002

T. Ruman et al. / Bioorganic Chemistry 38 (2010) 74–80
75
NMR chemical shifts, to assign thiophosphorylated protein NMR
resonances and prove the presence of thiophosphorylated lysine,
serine and histidine moieties.
All phospho and thiophospho derivatives of cysteine, serine and
lysine showed their resonances in the form of triplets, as an effect
of the three-bond proximity of two magnetically equivalent hydro-
3
gen atoms. The coupling constants JP–H for those systems are in
the 6–9 Hz range, in agreement with published data concerning
similar compounds [16,17].
2. Results and discussion
The most significant distinction between NMR properties of
phosphate and thiophosphate compounds concerns large differ-
ences between chemical shifts. While the 31P NMR resonances of
P-His are in the ꢀ7 to ꢀ10 ppm range, those of TP-His are in the
33–35 ppm region. The magnitude of those differences may reflect
a distinctly weaker electronic withdrawing effect of sulfur, as com-
pared with oxygen which causes strong electronic deshielding of
phosphorus. This downfield shifting effect is observable for all
phosphorylated and thiophosphorylated amino acid pairs, and is
especially visible for thiophosphocysteine, its two sulfur atoms
connected to phosphorus and 31P resonance shifted downfield to
54.2 ppm.
2.1. NMR spectra of phosphorylated and thiophosphorylated amino
acids
The 31P NMR results for side-chain P- and TP-derivatives are
presented in Table 1.
The most upfield shifted resonances (ꢀ5 to ꢀ10 ppm) among
investigated phosphorylated amino acids belongs to phosphohisti-
dines (P-His). Histidine reaction with phosphoramidates is signifi-
cantly faster than in case of other amino acids but P–N bond that
was created is vulnerable for acidic conditions as was found by
Boyer group [15]. Histidine reaction with phosphoramidate pro-
duces two isomers 1-phosphohistidine and 3-phosphohistidine,
the last one more thermodynamically stable. It should be noted
that P-His isomers differ significantly in their hydrolysis rate, with
1-phosphohistidine having a 5 min hydrolytic half-life at pH 2.4
(46 °C), while 3-phosphohistidine has approx. five times longer
half-life [13]. 31P NMR spectrum of histidine and phosphoramidate
post-reaction mixture contains two major resonances. The reso-
nances at upfield region seem to belong to 1- and 3-phosphorylated
histidine derivatives, with more upfield shifted resonance (ꢀ8 to
ꢀ10 ppm) belonging to 1-phosphohistidine (d1-phosphohistidine)
It should be noted that the averaged differences between 31P
NMR resonances of different pairs of P- and TP-amino acids are
comparable, ranging from 43.2 ppm difference with 1-P-/1-TP-
His, 43.4 ppm with P-/TP-Lys and 44.1 ppm with P-/TP-Ser to
41.0 ppm with 3-P-/3-TP-His and 38.3 ppm difference with P-/
TP-Cys. Thus knowing the mean difference value, roughly
42 ppm, may be useful in prediction of 31P NMR shifts of various
thiophosphates when chemical shifts of the corresponding phos-
phates are known.
and the other one (ꢀ6 and ꢀ7 ppm) to 3-phosphohistidine (e2
-
2.2. DFT calculations of 31P shieldings of TP-amino acids
phosphohistidine). 31P NMR spectrum contains also two small sin-
glet resonances belonging to 1,3-diphosphohistidine. The assign-
ment of NMR resonances was based on previously published data
[13].
With an accurate and reliable estimation of chemical shifts for
31P NMR method being still a long-term challenge, little is known
in this respect about thiophosphates and their derivatives. While
estimation of phosphorus chemical shift dP, based on comparison
of electronic shielding of particular atoms, might provide very use-
ful information regarding NMR properties of thiophosphates, it has
so far poorly described.
Phosphorus electronic shielding calculations of side-chain
thiophosphate derivatives of selected amino acids are presented
in Table 2. The TP-amino acid models used in these calculations
were divided into three groups based on molecular charge of mod-
el compound. All of the shielding values of TP-amino acids were
converted into chemical shift values using analogically obtained
The analogical reaction, carried out with thiophosphoramidate
instead of phosphoramidate, led to histidine thiophosphates that
are more hydrolytically stable than the corresponding phosphates,
thus having significantly longer half-life [15]. The 31P NMR
spectrum of thiophosphorylated histidine contained resonances
belonging to more thermodynamically stable 3-thiophosphohisti-
dine isomer (3-TP-His; at 34.2 ppm), less stable 1-thiophosphohis-
tidine (at 34.5 ppm) and two much smaller resonances belonging
most likely to the 1,3-dithiophosphate species (at 33.7 and
34.4 ppm).
Table 1
31P NMR chemical shifts of phosphorylated and thiophosphorylated amino acids.
P- or TP-amino acid
31P NMR chemical shift [ppm] and/multiplicity
Averaged P-/TP-amino acid shift difference [ppm]
P-Hisa
P-Arg
P-Lys
ꢀ5 to ꢀ10/s
–
–
–
5–6/s
0–0.2/tr
P-Cys
P-Ser
P-Tyr
15.9/tr
–
–
–
–
ꢀ0.3/tr
ꢀ3.8–0.0/s
P-Thr
ꢀ1.4/m
TP-Hisa
TP-Arg
TP-Lys
TP-Cys
TP-Ser
32.5–34.5/s
43.2 (1-P-His)e, 41.0 (3-P-His)e
–
–
43.4/tr
43.4f
38.3
44.1
–
54.2/tr
43.8/b
PSCl3
54.6/s, 49.7d; 28.6d; 15.7d; ꢀ0.1d/s
c
a
Histidine phosphorylation product contain 3-P-His, 1-P-His and diphosphate (with phosphate groups both at 1- and 3-positions) with relative integrals being 13.9:1:0.5
respectively. Also the thiophosphorylation product contain 3-TP-His, 1-TP-His and analogical dithiophosphate with relative integrals being 8.5:1:0.14.
b
Two doublets.
Common trace contaminant of thiophosphoramidates.
Phosphorus thiochloride hydrolysis products.
c
d
e
Calculated using d1-P-His = ꢀ8.7 ppm and d3-P-His = ꢀ6.8 ppm.
f
dP-Lys of 0.0 ppm was used for calculations (pH 7.5–7.8); higher values up to 0.2 ppm were observed at different pH values.

76
T. Ruman et al. / Bioorganic Chemistry 38 (2010) 74–80
Table 2
Phosphorus isotropic shielding constants (
r
P, ppm) of side-chain thiophosphorylated amino acids estimated with the B3LYP/aug-cc-pVTZ GIAO calculations. dP stands for 31P
chemical shift in relation to H3PO4, H2POꢀ or HPO42ꢀ
.
4
Neutral TP-amino acid
TP-Arg
TP-Cys
3-TP-His
TP-Lys
TP-Ser
TP-Thr
rP [ppm]
248.11
62.62
56.07
38.46
189.71
121.02
114.47
96.86
244.01
66.72
60.17
42.56
234.51
76.22
69.67
52.06
232.93
77.80
71.25
53.64
232.49
78.24
71.69
54.08
dP [ppm] rel. to H3PO4
dP [ppm] rel. to H2PO4ꢀ
dP [ppm] rel. to HPO42ꢀ
TP-amino acid monoanion
rP [ppm]
255.65
55.08
48.53
30.92
223.81
86.92
80.37
62.76
257.59
53.14
46.59
28.98
255.88
54.85
48.30
30.69
254.84
55.89
49.34
31.73
245.13
65.60
59.04
41.44
dP [ppm] rel. to H3PO4
dP [ppm] rel. to H2PO4ꢀ
dP [ppm] rel. to HPO42ꢀ
TP-amino acid dianion
rP [ppm]
276.55
34.17
27.63
10.02
216.67
94.06
87.51
69.90
255.33
55.40
48.85
31.24
266.40
44.33
37.78
20.17
256.36
54.37
47.82
30.21
244.65
66.08
59.53
41.92
dP [ppm] rel. to H3PO4
dP [ppm] rel. to H2PO4ꢀ
dP [ppm] rel. to HPO42ꢀ
TP-Cys ‘thiol’ form (P–SH) neutral
rP [ppm]
TP-Cys ‘thiol’ form (P–SH) monoanion
rP [ppm]
261.38
49.35
42.80
25.19
275.16
35.57
29.02
11.41
dP [ppm] rel. to H3PO4
dP [ppm] rel. to H3PO4
dP [ppm] rel. to H2PO4ꢀ
dP [ppm] rel. to H2PO4ꢀ
dP [ppm] rel. to HPO42ꢀ
dP [ppm] rel. to HPO42
values of reference compounds – neutral H3PO4, monoanionic
Ser, ꢀ0.30 ppm difference). These values are in a good agreement
with experimental ones that showed 31P NMR shifts of phosphates
and thiophosphates to have their resonances within the range of
almost 150 ppm. The computational prediction of 31P chemical
shift for TP-Arg and TP-Thr was also made (Table 2), suggesting
chemical shifts of 28.9 ppm for TP-Arg and 44.5 ppm for TP-Thr.
Of interest is that the chemical shift values of TP-Cys phospho-
rus (see Table 2), as compared with those for TP-His, TP-Ser and TP-
Lys, are much higher, although calculated in an identical manner.
Furthermore, this value (87.3 ppm) differs considerably from
experimental one (54.2 ppm). The explanation of this phenomenon
was described previously by our research group [18]. The calcula-
tion results strongly suggest that TP-cysteine is existing in equilib-
rium of ‘thione’ tautomeric form with P@S formal double bond
along with ‘thiol’ form with P@O formal double bond and ionized
or protonated sulfur P–Sꢀ/P–SH. Those ‘thiol’ tautomeric forms
are having their 31P NMR resonances upfield shifted by a factor of
20–30 ppm [18]. In order to estimate the chemical shift of ‘thiol’
form, we have conducted another set of calculations and those re-
sults are presented in Table 2 (bottom part). The calculated phos-
phorus chemical shift of neutral form of ‘thiol’ TP-Cys (in relation
to H3PO4, with 33.7 ppm correlation factor) is approx. 16.7 ppm
H2POꢀ and HPO24ꢀ dianion using the following equation:
4
dP ¼ rref
ꢀ rx ðRef: ½15ꢁÞ
where dP is phosphorus NMR chemical shift, rref – isotropic phos-
phorus shielding of reference compound and rx
– isotropic
phosphorus shielding of analyzed compound.
It should be noted that precise estimation of phosphorus NMR
chemical shift is still an unsolved and very difficult problem be-
cause of (i) unreliable estimation of concentration of neutral,
mono- and dianionic forms in solution, (ii) complexity of interac-
tions between analyzed compounds and buffer ingredients and
also (iii) calculations of shieldings of those mentioned forms being
still inaccurate. In our calculations the best match of obtained shift
values with experimental ones was achieved with neutral TP-ami-
no acid models (Table 2, Fig. 1). The experimental values of 31P
NMR chemical shifts for 3-TP-His (32.5 ppm), TP-Lys (43.4 ppm)
and TP-Ser (43.8 ppm) could be correlated with neutral TP-amino
acid models (related to H3PO4) by subtracting an additional factor
of 33.7 ppm value. This subtraction yields the following values of
correlated chemical shifts: 33.0 ppm (TP-His, ꢀ0.4 ppm differ-
ence), 42.5 ppm (TP-Lys, 0.88 ppm difference) and 44.1 ppm (TP-
N
N
OH
OH
N
OH
N
N
P X
OH
N
P X
OH
S
P X
OH
P-Cys
P-Arg
3-P-His
OH
P X
OH
OH
OH
P X
OH
NH
O
P X
OH
O
P-Ser
P-Lys
P-Thr
Fig. 1. Structures of the models of phosphorylated and thiophosphorylated amino acids. The symbol X denotes O or S atom.

T. Ruman et al. / Bioorganic Chemistry 38 (2010) 74–80
77
being very similar to previously described thiophosphate ‘thiol’
forms [18]. The almost equimolar equilibrium of ‘thione’
(87.3 ppm) and ‘thiol’ (16.7 ppm) forms should give observed value
of 54.2 ppm.
Ser thesis is strongly supported by an almost identical chemical
shift of TP-serine obtained by direct thiophosphorylation of serine
(see Table 1) and 31P–1H gradient enhanced HMBC spectra pointing
to 3.6 ppm as 1H chemical shift of phosphorus-coupled hydrogen
atoms.
Two triplet resonances at approx. 43.3 and 43.4 ppm corre-
spond apparently to TP-Lys as thiophosphorylation of free amino
acid lysine gave a very similar result (Table 1). The 31P NMR spec-
trum of TP-Lys that was measured at identical pH as thiophosph-
2.3. Thiophosphorylation of Caenorhabditis elegans thymidylate
synthase
Fig. 2 presents thiophosphate ‘fingerprint’ region of 31P NMR
spectrum. Spectrum B at Fig. 2 presents results of the thiophosph-
orylation of Tris–HCl buffer, identical to one used for modification
of thymidylate synthase. The thiophosphorylated Tris (fragment B
at Fig. 2) presents two multiplets at approx. 42.3 and 43.9 ppm that
are also observable in thiophosphorylated protein NMR spectrum
(spectrum C at Fig. 2). The resonances of thiophosphorylated Tris
at 42.3, 43.9 ppm (JP–H = 7.6 Hz and 6.6 Hz respectively) are triplets
as a result of heteronuclear coupling of thiophosphate phosphorus
with two magnetically equivalent hydrogen atoms from neighbor-
ing methylene group [16]. Spectrum A in Fig. 2 presents the above
described fingerprint region of thiophosphorylated protein with
H-decoupling applied, showing clearly that thiophosphorylated
Tris resonances are singlets, thus proving the three-bond P–H
couplings.
orylated thymidylate synthase sample, contains
a
triplet
resonance at 43.4 ppm. Also, the 31P–1H HMBC spectra confirm
that phosphorus-coupling hydrogen atoms have 1H NMR shifts of
approx. 3.1 ppm, corresponding to e-CH2 group of lysine moiety.
Another interesting observation that stems from the 2D spectrum
is that the phosphorus atoms of both triplet resonances in TP-ly-
sine region (43.3–43.4 ppm) are coupled to exactly the same 1H
chemical shift (3.1 ppm), suggesting those two triplets to reflect
two forms of protein TP-lysine moiety resulting probably from a
slow, at NMR-scale, protonation/deuteration of lysine
atom caused by bulky thiophosphate substituent.
e-nitrogen
The results of our studies on free amino acid phosphorylation
and thiophosphorylation pointed to histidine being the fastest
reacting amino acid in the phosphorylation and thiophosphoryla-
tion reactions (not shown). Histidine thiophosphorylation reac-
tions were performed with the free amino acid, as well as C.
elegans recombinant thymidylate synthase protein (see Figs. 3
and 4, Table 1).
The thiophosphorylated thymidylate synthase NMR spectrum is
presented in Fig. 2, part C. Besides the thiophosphorylated Tris res-
onances, two triplets are present at approx. 43.2 and 43.3 ppm
(coupling constants JP–H = 6.5 and 5.6 Hz respectively) and two
doublets at 43.8 ppm (JP–H = 5.7 and 8.5 Hz). Coupling constants
mentioned above are in the range typical for three-bond P–H cou-
pling [19]. All of the resonances from thiophosphorylated protein
are singlets with H-decoupling applied (Fig. 2, spectrum A), prov-
ing the three-bond proximity of thiophosphate phosphorus to the
methylene hydrogen atoms. The resonance at approx. 43.8 ppm
apparently belongs to the thiophosphorylated serine side-chain
hydroxyl. The 31P resonance of newly formed TP-Ser is in the form
of two doublets, resulting from the heteronuclear coupling with
phosphorus of two magnetically nonequivalent (shift difference
0.04 ppm) hydrogen atoms from TP-serine moiety methylene
group. Those two magnetically nonequivalent hydrogen atoms
The free amino acid histidine thiophosphorylation reaction was
carried out in ammonium carbonate buffer (pH 7.4). Series of res-
onances were found at 32.5–34.5 ppm region that is very near to
that from an older work of Pirrung group showing approx
35 ppm chemical shift of TP-His [13]. However, that spectrum
was obtained in deuterium oxide and no exact pH or pD value were
presented; it should be noted that 31P NMR chemical shifts of ions
significantly depend on pH (or pD). The 31P NMR spectrum of
thiophosphorylated thymidylate synthase also presents two sin-
glet resonances at 32.5 and 32.7 ppm laying in the TP-His region
(Fig. 3). The more downfield shifted resonance has relative integral
of 1.0 with another upfield shifted one of 1.7. The assignment of
those two resonances was made by analysis of free histidine
thiophosphorylation NMR results and limited literature data
[13,15]. It seems that more upfield shifted resonance belongs to
hydrolytically more stable 3-thiophosphohistidine (3-TP-His) and
another one downfield shifted resonance from less hydrolytically
stable 1-thiophosphohistidine (1-TP-His).
The exceptionally interesting part of the spectrum of
thiophosphorylated thymidylate synthase is presented in Fig. 4.
There are two singlet resonances at ꢀ6.2 and ꢀ6.3 ppm laying in
region characteristic for phosphohistidines (P-His). Our free amino
acid hisitidine phosphorylation results confirm that those two sin-
glet resonances almost certainly belong to the 1-P-His and 3-P-His
being most likely a hydrolysis product of 1-TP-His and 3-TP-His
respectively. Relative integrals measured for those two resonances
3
have very similar coupling constants JP–H = 5.65 and 5.67 Hz. TP-
A
B
TP-his
C
33.5
33.0
32.5
δ [ppm]
32.0
31.5
44.0
43.5
43.0
42.5
42.0
Fig. 2. Thiophosphate fingerprint region of 31P NMR spectrum of thymidylate
synthase thiophosphorylation product with 31P–1H decoupling (A) and without H-
decoupling (C). Spectrum B shows thiophosphorylation product of Tris–HCl buffer.
Fig. 3. Thiophosphohistidine resonances of 31P NMR spectrum of thiophosphory-
lated thymidylate synthase.

78
T. Ruman et al. / Bioorganic Chemistry 38 (2010) 74–80
tive environment. The NMR spectra of obtained modified proteins
Pi
were assigned, based on NMR spectra of thiophosphorylated amino
acid samples and the computational (DFT) estimation of 31P NMR
chemical shifts, indicating the presence of thiophosphorylated
lysine, serine and histidine moieties. Several methods presented
here should be useful for prediction of 31P NMR chemical shifts
of thiophosphorylated amino acid moieties and even thiophos-
phates in general.
4. Experimental
P-his
4.1. Materials and methods
Cellulose dialysis tubing, DE52-Cellulose, O-phosphotyrosine
and O-phosphothreonine were purchased from Sigma–Aldrich.
Diammonium- and potassium phosphoramidates (diammonium
TPA and KTPA respectively) were prepared according to Pirrung
et al. (2000).
3
2
1
0
-1
-2
-3
-4
-5
-6
-7
δ [ppm]
Fig. 4. Phosphohistidine resonances of 31P NMR spectrum of thiophosphorylated
thymidylate synthase. Pi = inorganic phosphate.
4.2. NMR analysis
All NMR spectra were obtained with Bruker Avance spectrome-
ter operating in the quadrature mode at 500.13 MHz for 1H and
202.46 MHz for 31P nuclei. The residual peaks of deuterated sol-
vents were used as internal standards in 1H NMR method. 31P
NMR spectra were recorded at 277 K both with and without proton
decoupling. The internal standard used in 31P NMR was inorganic
phosphate (Pi) having its resonance at 2.15 ppm (at pH 7.8),
2.05 ppm (pH 7.5), 1.65 ppm (at pH 5.0) and 0.0 ppm (at pH 1.5).
All samples were analyzed using gradient enhanced 1H–31P Heter-
onuclear Multiple Bond Correlation (HMBC) experiments. The
HMBC experiments were optimized for long range couplings by
are 1:2.4 for 1-P-His and 3-P-His respectively. Thiophosphate may
hydrolyze to phosphate in aqueous solutions, as evidenced by the
time-dependent growth of the inorganic phosphate resonance at
approx 2.2–2.4 ppm (see Fig. 4).
The enzyme thiophosphorylation was carried out also in ammo-
nium carbonate buffer, pH 7.4, with the use of diammonium thi-
ophosphoramidate. The results were similar to those obtained
with Tris buffer but for (i) much lower total amount of thiophos-
phohistidines, (ii) higher 3-TP-His/1-TP-His molar ratio and (iii)
much smaller corresponding resonances from TP-Ser and TP-Lys.
The mass spectrometry was also used to propose the location of
the thiophosphate moiety in the thymidylate synthase molecule.
The preliminary results obtained from trypsin digestion of enzyme
shows peak at m/z 1825.805 The experimental isotopic patterns of
the positively charged ion are in perfect agreement with the simu-
lated isotopic pattern distributions for monothiophosphate of
TVQQQVHLNQDEYK where thiophosphate moiety is attached to
histidine (His26) or lysine (Lys33) side-chain (Fig. 1 in supplemen-
tary material).
Potential influence of the thiophosphorylation on thymidylate
synthase activity was tested with recombinant human and C. ele-
gans preparations of the enzyme. Each preparation underwent thi-
ophosphorylation with diammonium TPA in Tris–HCl buffer, pH
7.5, at 4 °C under conditions described in Material and methods,
with the enzyme activity determined in samples of the reaction
mixture taken just after addition of diammonium TPA, and at sev-
eral time points during 24 h period. Separate samples were incu-
bated under the same conditions, but without diammonium TPA
added, in order to test the enzyme stability. The results indicated
the enzyme activity to be lowered, following 24 h reaction, to
ꢂ50% of the control value due to the thiophosphorylation process
alone, pointing to a possibility that the modification influences
the enzyme-catalyzed reaction parameters, as reported for phos-
phorylation [14].
3
using different JP–H values (1–20 Hz). The 1H NMR spectra were
obtained with the use of HDO suppression method. All buffer solu-
tions used for NMR spectroscopy were based on deuterium oxide
of 100%D purity (Armar Chemicals AG).
4.3. Mass spectrometry
The mass spectrum was obtained on a Bruker MicrOTOF-Q
spectrometer (Bruker Daltonik, Bremen, Germany), equipped
with Apollo II electrospray ionization source with ion funnel.
The mass spectrometer was operated in the positive ion mode.
The instrumental parameters were as follows: scan range m/z
200–2200, dry gas–nitrogen, temperature 180 °C, ion source volt-
age 4500 V, reflector voltage 1300 V, detector voltage 1920 V.
The instrument was calibrated externally with the Tunemix™
mixture (Bruker Daltonik, Germany) in quadratic regression
mode. The sample (0.1 M Tris–HCl buffer of pH 7.5 containing
approx. 50
and thiophosphate salts) was diluted twice with methanol and
infused at a flow rate of 3 l/min. The mass spectrum was
lg of protein and traces of thiophosphoramidate
l
deconvoluted using the maximum entropy method.
The protein (0.2 mg) digestion was performed in the 0.2 M Tris–
HCl buffer (pH 7.5) by addition of trypsin (10 lg) the reaction mix-
ture was incubated at 24° C overnight. The solution was desalted
on C18 Sep-pak Plus Cartridges (Waters, Ireland). Washed with
water (10 ml) and eluted with acetonitrile (0.7 ml). The solution
was concentrated in vacuo and introduced to mass spectrometer.
Analysis of the obtained spectra was performed with the Bruker
Daltonics Compass DataAnalysis v.4 software The mass accuracy
together with the isotopic pattern (using SmartFormula) enabled
an unambiguous confirmation of the elemental composition of
the obtained tryptic peptides.
3. Conclusions
The thiophosphorylation reactions of recombinant thymidylate
synthase protein were performed using potassium thiophosph-
oramidate and diammonium thiophosphoramidate salts in Tris-
and ammonium carbonate based buffer solutions at non-destruc-

T. Ruman et al. / Bioorganic Chemistry 38 (2010) 74–80
79
4.4. Calculations
4.10. Thiophosphorylation of thymidylate synthase with diammonium
TPA with buffer exchange
The theoretical calculations have been performed with the den-
sity functional B3LYP/aug-cc-pVTZ method. To save computational
time, the structures of phosphorylated and thiophosphorylated ami-
no acids were truncated by removing some groups of atoms remote
from the phosphorylated regions (Fig. 1). The optimal geometries
were obtained and confirmed with positive harmonic frequencies,
then NMR shieldings for models of phosphorylated and thiophosph-
orylated amino acids in their neutral, monoanionic and dianionic
forms were calculated. All the calculations were performed with
the Gaussian G03 (rev. C.02) suite of programs [20].
To the protein buffer solution (1 ml of 0.2 M Tris–HCl, pH 7.5
containing 2.1 mg of protein) diammonium TPA (23.0 mg) was
added and shaken for 7 h at 277 K. Dialysis of the post-reaction
mixture was then conducted by introducing obtained mixture into
dialysis bag and dialyzed four times in ammonium carbonate buf-
fer (4 ꢃ 300 ml of 0.1 M ammonium carbonate buffer, pH 7.4).
NMR sample contained 0.5 ml of dialysis product and 0.3 ml of
deuterium oxide.
4.11. Thiophosphorylation of thymidylate synthase with diammonium
TPA in ammonium carbonate buffer
4.5. Thiophosphorylation of amino acids ( -histidine,
L
L-arginine,
L
-lysine, -cysteine, -serine, -proline and L-tyrosine)
L
L
L
Diammonium TPA (13.4 mg) was introduced into protein solu-
tion (840 lg of C. elegans thymidylate synthase in 0.1 M ammo-
nium carbonate buffer pH 7.4).
Ammonium carbonate buffer solution of amino acid (0.2 mmol
of amino acid in 500 l of buffer at pH 7.4) was reacted with diam-
l
monium thiophosphoramidate (1:3 amino acid to diammonium
TPA molar ratios). Reaction mixture was shaken for 72 h at 277–
Acknowledgments
278 K temperature. NMR sample was prepared by mixing 400 ll
Supported by the Ministry of Science and Higher Education of
Poland, Grant Nos. N401 024036 and N204 088 31/2052. The
ICM computer center, University of Warsaw, Poland, is acknowl-
edged for the G18-6 computer grant.
of post-reaction mixture with 300
ll of deuterium oxide. NMR re-
sults are presented in Table 1.
4.6. Phosphorylation of amino acids (
L-histidine,
L-arginine, L-lysine, L-
cysteine, -serine, -proline, -tyrosine and L
L
L
L
-threonine)
Appendix A. Supplementary data
Water solution of amino acid (0.2 mmol of amino acid in 500 ll
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bioorg.2009.11.002.
of water) was reacted with potassium phosphoramidate (1–10
amino acid to KPA molar ratios). Reaction mixture was shaken
for 48 h at 277–278 K temperature. NMR sample was prepared
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80
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