Synthesis, characterization and application of two nucleoside
triphosphate analogues, GTPcNH
2
and GTPcF
Michael Stumber
1
, Christian Herrmann
2
, Sabine Wohlgemuth
2
, Hans Robert Kalbitzer
1
, Werner Jahn
1
and Matthias Geyer
1,
*
1
Max-Planck-Institut fu
¨
r medizinische Forschung, Department of Biophysics, 69120 Heidelberg, Germany;
2
Max-Planck-Institut fu
¨
r molekulare Physiologie, Department of Structural Biology, 44227 Dortmund, Germany
Guanosine triphosphate nucleotide analogues such as
GppNHp (also named GMPPNP) or GTPcSarewidely
used to stabilize rapidly hydrolyzing protein-nucleotide
complexes and to investigate biochemical reaction path-
ways. Here we describe the chemical synthesis of guanosine
5¢-O-(c-amidotriphosphate) (GTPcNH

)1
,similar
to that for the natural substrate GTP. For GTPcF we ob-
tained a similar enthalpy of DH° ¼ 3.9 kcalÆmol
)1
while the
magnesium association constant is only K
a
¼ 0.2 m
M
)1
.The
application of both guanine nucleotide analogues to
the GTP-binding protein Ras was investigated. The rate of
hydrolysis of GTPcNH
2
bound to Ras protein lay between
the rates found for Ras-bound GTPcS and GppNHp, while
Ras-catalysed hydrolysis of GTPcF was almost as fast as for
GTP. The two compounds extend the variety of nucleotide
analogues and may prove useful in structural, kinetic and
cellular studies.
Keywords: nucleotides; nucleotide analogues; NMR spectro-
scopy; GTP hydrolysis; Ras.
Nucleotides are fundamental components in cellular meta-
bolism. Acting as substrates for nucleotide binding proteins,
they are the protagonists of a large variety of cellular
processes. Nucleotides can regulate enzymatic activity by
transitions between their mono-, di- and triphosphate
bound forms. These transitions often induce conformational

2
p
(also named GMPPCP) and their respective adenosine
counterparts AppNHp and AppCH
2
p. Another application
of substrate analogues is the use of caged nucleotides to
characterize unstable protein intermediates by X-ray crys-
tallography [4]. Nucleotide modifications can also serve as
an approach to designing dominant negative forms of a
protein [5] or to solve the phase problem in crystallography
[6]. Even more specific is the application of aluminium
fluoride, beryllium fluoride or orthovanadate in the presence
Correspondence to M. Geyer, Max-Planck-Institut fu
¨
r medizinische
Forschung, Abteilung Biophysik, Jahnstraße 29,
D-69120 Heidelberg, Germany.
Fax: + 49 6221 486 437, Tel.: + 49 6221 486 396,
E-mail: [email protected]
Abbreviations:GTPcNH
2
, guanosine 5¢-O-(c-amidotriphosphate);
GTPcF, guanosine 5¢-O-(c-fluorotriphosphate); GppNHp, guanosine
5¢-O-(b,c-imidotriphosphate); GppCH
2
p, guanosine 5¢-O-
(b,c-methylenetriphosphate); GTPcS, guanosine 5¢-O-(c-thiotriphos-
phate); ITC, isothermal titration calorimetry; DCC, dicyclohexylcar-
bodiimide; DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate;

guanosine 5¢-O-(c-amidotriphosphate) (GTPcNH
2
), for
which we describe the first synthesis, and guanosine 5¢-O-
(c-fluorotriphosphate) (GTPcF) [9], which we synthesized
by the method of Wittmann [10]. Both are shown in Fig. 1.
We characterized the stability and metal ion binding
properties of the two nucleotide analogues by NMR
spectroscopy and isothermal titration calorimetry. Both
nucleotides were bound to the small GTP-binding protein
Ras and the rates of hydrolysis were determined in
comparision to other nucleotide triphosphate derivatives.
Finally, the suitability for spectroscopic and structural
studies was tested by formation of the complex between
RasGTPcNH
2
and the Ras-binding domain of the Ras
effector protein c-Raf-1.
MATERIALS AND METHODS
General description of synthesis
High pressure liquid chromatography (HPLC) was done on
a Beckman ÔSystem GoldÒÕ. Nucleotides were analysed by
ion-pair chromatography on a reversed phase Super ODS
column, 50 · 4.6 mm (TOYOPEARLÒ) at a flow rate of
1.2 mLÆmin
)1
, using a linear gradient from 100% 10 m
M
tetrabutyl-ammonium bromide/10 m
M

ture the mixture was treated with about 5 mL concentrated
ammonia in water for 30 min. The solution was diluted with
60–70 mL water and, after filtration, applied to a Super Q
column (2.5 · 20 cm). The column was eluted at a rate of
5mLÆmin
)1
with a gradient from 0 to 1
M
THC within
120 min. Fractions containing the GTPcNH
2
(as checked
by UV absorption and HPLC, retention time 4.20 min)
were collected and evaporated. Any remaining THC was
removed by dissolving in methanol and repeated evapor-
ation under reduced pressure, yield of the pure GTPcNH
2
was 50–60%.
One gram of GTP triethylammonium salt was added to a
stirred solution of 2.5 mL tributylamine and 1.2 g
2,4-dinitrofluorobenzene in about 10 mL dimethylforma-
mide. After 6–8 h a clear solution was obtained. The
mixture was kept for 20–24 h at room temperature. The
crude product was precipitated with 100 mL acetone and
300 mL diethyl ether. The pellet was dissolved in water
(about 20 mL) and applied to a Super Q column
(18 · 2.6 cm). The column was eluted with a gradient of
0–1
M
THC within 2 h at a flow rate of 5 mLÆmin

2
O/D
2
O. Typically the
lyophilized nucleotide was redissolved to a final concentra-
tion of 2–10 m
M
and 2500 lL of the sample volume was
placed in 10 mm NMR tubes (Wilmad). For titration
experiments various amounts of MgCl
2
were added from a
100-m
M
stock solution. Proton NMR experiments were
performed using 500 lL sample volume in 5 mm NMR
tubes (Wilmad).
31
P NMR spectra of C-terminal truncated
wildtype Ras protein (residues 1–167) complexed with
GTPcNH
2
ÆMg
2+
were recorded in 40 m
M
Tris/HCl, 5 m
M
MgCl
2

P NMR spectra of free nucleotides,
64–512 free induction decays were summed after excitation
with a 65 degree pulse using a repetition time of 3–5 s. A
total of 32 K time domain data points were recorded and
transformed to 16 K real data points corresponding to a
digital resolution of 0.74 Hz point
)1
.The
31
P spin-spin
coupling constants of the nucleotide-Mg
2+
complexes were
determined from a nonfiltered 1D spectrum with a digital
resolution of 0.25 Hz per point after Fourier transforma-
tion.
All spectra were processed on a Silicon Graphics Indigo2
workstation using the software package UXNMR (Bruker,
Karlsruhe) for data processing and data evaluation. Phos-
phorus spectra used for exchange rate determination were
filtered by an exponential window function causing no
significant line broadening.
Determination of exchange rates
The Mg
2+
exchange rates of GTPcNH
2
were extracted
from a series of
31

P
spectra (a-, b- and c-phosphorus nuclei), using chemical
shift values and J-coupling constants as listed in Tables 1
and 2.
Isothermal titration calorimetry
The interaction between a nucleotide and the magnesium
ion was investigated by means of ITC (ITC-MCS, Micro-
Cal, Inc.). Briefly, in such an apparatus the solutions are
thermostatted to the desired temperature, the nucleotide at
5.0 m
M
placed in a cell which is accurately temperature
controlled and the MgCl
2
solution at 50 m
M
in a syringe
dipping into the cell. The two solutions are mixed by
computer controlled stepwise injections (typically in inter-
vals of 4 min) from the syringe which serves at the same
time as a stirrer. The heat consumed due the endothermic
association process is measured by the detection of the
heating power which is necessary to keep the cell at constant
temperature [15]. All ITC experiments were performed at
25 °C. The data were analyzed using the manufacturer’s
software yielding the stoichiometry N, the binary equili-
brium association constant K
a
¼ [nucleotideÆMg
2+

GTPcNH
2
)11.46 )22.76 )1.12 –
GTPcNH
2
ÆMg
2+
)11.33 )21.50 )0.34 –
GTPcF )11.66 )23.49 )18.16 0.91
GTPcÆFMg
2+
)11.89 )23.19 )18.63 0.97
GTP )10.74 )21.22 )5.51 –
GTPÆMg
2+
)10.41 )19.01 )5.30 –
Table 2. J-Coupling constants of GTPcNH
2
and GTPcFinaqueous
solution.
J-coupling constants (Hz)
Nucleotide
2
J
PaPb
2
J
PbPc
1
J

reagents were purchased from Sigma and GppCH
2
pwas
ordered from JenaBioScience. GDP, which binds very
tightly to Ras, was replaced with the respective GTP
analogue by the following procedures. For nucleotide
exchange GTPcNH
2
, GppNHp and GppCH
2
pwereeach
incubated at threefold molar excess with Ras in the presence
of 200 l
M
ammonium sulfate, 0.1 l
M
zinc chloride and 1 U
alkaline phosphatase per mg Ras overnight at 4 °C. In order
to load Ras with GTP, GTPcF, or GTPcS nucleotide-free
Ras was produced by incubation overnight at 4 °Cinthe
presence of 200 l
M
ammonium sulfate, 0.1 l
M
zinc chloride
and 0.2 U alkaline phosphatase per mg Ras. After size
exclusion chromatography, one of the nucleotides was then
added to the Ras protein. Excess nucleotide after either
procedure was removed (which is important in order to
obtain accurate single turnover hydrolysis rate constants).

H NMR measurements of both GTPcNH
2
and GTPcF in aqueous solution at 20 °C, pH 7.4 showed
no difference to the natural substrate GTP [19] as the
guanine base is not affected by the modifications and as
the c-phosphate amide hydrogens are in fast exchange with
the solvent. In Fig. 2
31
P NMR spectra are shown for
GTPcNH
2
and GTPcF, and their respective metal ion
complexes with Mg
2+
.
For GTPcNH
2
the appearance of three discrete reson-
ance lines with similar intensity confirms the uniformity and
the conformational identity of the substrate. The observed
mean half width of, e.g. 4.7 Hz for the c-resonance line is
typical for a molecule of 523 Da mass at 20 °C in aqueous
solution. The resonance lines could be assigned by their
J-coupling constants and by comparison to unmodified
GTP. While the chemical shift of the a-phosphate group
changed only little upfield compared to GTP, the
b-phosphate was shifted upfield by about )1.5 p.p.m. and
the terminal c-phosphate shifted by almost 5 p.p.m. down-
field by the replacement of the hydroxy OH
–

b
and P
b
–P
c
were almost identical, while coordination
with magnesium again decreased the coupling constants.
In GTPcF four phosphorus lines appeared as the
coupling between the natural spin ½ nuclei
31
Pand
19
F
led to a splitting of the terminal phosphate resonance. This
direct coupling constant
1
J
PcF
was about 936 Hz and hardly
changed upon magnesium coordination (934 Hz), indica-
ting a strong interaction between the two nuclei. Chemical
shift changes of GTPcF compared to GTP were much more
distinct than for GTPcNH
2
. All three phosphate groups
shifted upfield; in the case of the c-phosphate the shift was
)12.6 p.p.m. By contrast, coordination to magnesium
caused only slight chemical shift changes, of which the
Fig. 2.
31

perature was less than 1% in five days. In contrast, at
pH 4.5 in 0.1
M
potassium phosphate buffer the nucleotide
was hydrolysed to GDP with a half time of about 48 h.
Titration of GTPcNH
2
with HCl/NaOH monitored by
31
P
NMR spectroscopy showed no variation of the chemical
shifts of the three-fold negatively charged phosphate groups
from pH 3 to pH 11. At pH 2.8 the intrinsic hydrolysis
increased (so called acidic hydrolysis) and GTPcNH
2
3–
was
rapidly transformed to GDP
3–
+H
2
PO
4
–
+NH
4
+
by two
water molecules. The intermediate compound phosphor-
acid-amidate H

3
NH
2
]
2–
at +7.97 p.p.m. The pK
a
values between these
three states were determined to pK
(0/1–)
¼ 3.02 ± 0.05 and
pK
(1–/2–)
¼ 8.46 ± 0.02 using a least square fit to 15
individual measured chemical shift values (data not shown).
Since the resonancelinesforthe a- and b-phosphate groups of
GDP at pH 2.8 were located at )10.73 and )10.20 p.p.m.,
respectively, a possible signal overlap between GTPcNH
2
,
GDP, HPO
3
NH
2
and H
3
PO
4
(P
i

2
to
magnesium by a complete lineshape analysis of a series of
NMR spectra. We adjusted the saturation of GTPcNH
2
with Mg
2+
to 45% and varied the temperature from 5 °Cto
65 °C in 13 steps of 5°. Five representative
31
P NMR spectra
of the b-resonance line and the corresponding simulations
are shown (Fig. 4). The fitted exchange rates in aqueous
solutions ranged from 900 to 9000 Hz with relative margins
from ± 22% at 5 °C to ± 8% at 30 °C. As the plot
against reciprocal temperature shows, the simulated
exchange rates k nicely fit to the Arrhenius equation
k ¼ k
0
exp(–DH
à
/RT) with R the gas constant and T the
absolute temperature (Fig. 5). Based on these values the
activation energy DH
à
for the GTPcNH
2
Mg
2+
complex

negative charge at the c-position in GTPcNH
2
and GTPcF
Fig. 3.
31
PNMRspectraofaMgCl
2
titration series added in increasing
amount to GTPcNH
2
. The resonance line of the b-phosphate in the
experimental measurements (left) and its corresponding simulation
(right) are shown. Note the shift and the intermediate broadening of
the resonance line. The amount of GTPcNH
2
Mg
2+
complexes relative
to free GTPcNH
2
nucleotide is indicated on the left. The determined
exchange rates based on the exchanging spin system simulation are
shown right. The spectra were measured at 20 °C and pH 7.4 in
aqueous solution.
3274 M. Stumber et al. (Eur. J. Biochem. 269) Ó FEBS 2002
where the protic hydroxy group is replaced by the amino
and fluoride groups, respectively. In contrast, the sulfur in
GTPcS may take on the role of the oxo-group. It should be
noted that the smaller affinities of GTPcNH
2

.
Fig. 6. Isothermal titration calorimetry of GTPcNH
2
with MgCl
2
. To a
solution of 5.0 m
M
GTPcNH
2
placed in the cell of the calorimeter a
solution of 50 m
M
MgCl
2
wasinjectedinstepsof6lL each (the first
step was 2 lL only). The increase in heating power was detected (upper
panel). The power pulses were integrated and plotted vs. the molar
ratio of injected MgCl
2
and nucleotide (lower panel). A fit to the
experimental data yields the stoichiometry factor N ¼ 0.96, the
association constant K
a
¼ 0.82 m
M
)1
and the enthalpy of association
DH° ¼ 3.9 kcalÆmol
)1

GTPcF 0.84 0.20 3.9 24
Fig. 4.
31
P NMR spectra of a temperature series of GTPcNH
2
Mg
2+
.
The b-phosphate resonance line at )22.19 p.p.m. is shown in an
intermediate exchange state at 45% Mg
2+
saturation. Experimental
measurements (left) and simulated spectra (right) are displayed
showing temperature values and the simulated exchange rates,
respectively. Lyophilized GTPcNH
2
was dissolved to 2.1 m
M
con-
centration in aqueous solution and adjusted to pH 7.4 with HCl/
NaOH. MgCl
2
was added to 1 m
M
concentration. The precise
saturation was determined from the chemical shift position at 20 °C
(see Fig. 3 and Table 1).
Ó FEBS 2002 Nucleotide analogues GTPcNH
2
and GTPcF(Eur. J. Biochem. 269) 3275

GTPcNH
2
. At low temperature (5 °C) the b-phosphate
resonance was split into a less populated high field shifted
peak (b1,  27%) and a highly populated down field shifted
peak (b2,  73%). A temperature series from 2 °Cto30°C
revealed the coalescence of both lines at approximately
15 °C which is typical for a two-site exchange with a
transition from slow to fast exchange (data not shown). As
described for the intrinsic hydrolysis of GTPcNH
2
,theRas-
catalysed hydrolysis of bound GTPcNH
2
did not lead to the
observable formation of the compound H
2
PO
3
NH
2
in the
NMR spectra (which is expected at )2.7 p.p.m.). Instead,
the resonance signals for P
i
and Ras-bound GDP increased
during the time course of the experiment (Fig. 7, compare
bottom and top spectra) suggesting the formation of
ammonia and Ras-bound GTP before hydrolysis.
A concentration series with the effector protein Raf-RBD

2+
protein
at 37 °C bound to various guanosine triphosphate nucleotide analogues.
Hydrolysis rates were determined with HPLC by measuring the con-
centration of protein-bound tri- and diphosphate nucleotides. Buffer
conditions: 25 m
M
Tris/HCl at pH 7.4, 2.5 m
M
MgCl
2
and 1 m
M
DTE.
Nucleotide GTPase rate (10
)5
min
)1
)
RasGTP 2820
RasGTPcF 1427
RasGTPcS 252
RasGTPcNH
2
84.6
RasGppNHp 25.6
RasGppCH
2
p 15.0
Fig. 7.

31
P chemical shifts of Ras
(1)167)
Æ
GTPcNH
2
Æ
Mg
2+
at pH 7.4,
5 °C. Spectra were recorded in 25 m
M
Tris/HCl buffer, 2.5 m
M
Mg
2+
and 1 m
M
DTE. The splitting of the a- and b-phosphate resonance
lines in protein bound triphosphate-nucleotides is a specific feature of
the Ras protein, indicating different conformations of the active center
[24].
31
P chemical shift (p.p.m.)
Proteinnucleotide a
(1)
a
(2)
b
(1)

a
for magnesium is significantly
smaller for both nucleotides analysed. Therefore, the more
stable c-amido triphosphate analogue may be particularly
useful for the study of the role of divalent cation binding,
e.g. to analyse a proposed reaction mechanism. For
example, the influence of the magnesium binding affinity
on the kinetics of the Ras guanine nucleotide exchange
factor Sos [36] can be studied with the nucleoside
diphosphate GDPcNH
2
derivative loaded onto Ras.
Additionally, an intermediate magnesium-free state may
be stabilized more easily with GTPcNH
2
or ATPcNH
2
analogues. The analogue ATPcNH
2
may provide new
insights into the equilibrium between different conforma-
tions of myosin [37]. Finally, specific labeling of the amide
group with
15
N isotopes may be useful for nitrogen
selective heteronuclear NOE experiments for the structural
analysis of the active center in solution. In combination
with specific labeling of single residues in the protein [38]
this may yield detailed insights into the dynamics of the
nucleotide binding site.

function of the protein. This is particularly important for
mechanistic studies and the analysis of binding intermedi-
ates, as shown for the complex formation of Ras with the
effector protein Raf-RBD [24,41]. With these two deriv-
atives characterized we extend the variety of nucleotide
analogues available for kinetic, structural, and cellular
studies.
ACKNOWLEDGEMENTS
We thank John Wray and Roger S. Goody for discussions and Ulrich
Haeberlen, Alfred Wittinghofer and Kenneth C. Holmes for continu-
ous support. M.G. acknowledges support by the Peter und Traudl
Engelhorn Stiftung (Penzberg, Germany).
REFERENCES
1. Bar-Sagi, D. & Hall, A. (2000) Ras and Rho GTPases: a family
reunion. Cell 103, 227–238.
2. Nakielny, S. & Dreyfuss, G. (1999) Transport of proteins and
RNAs in and out of the nucleus. Cell 99, 677–690.
3. Zerial, M. & McBride, H. (2001) Rab proteins as membrane
organizers. Nat. Rev. Mol. Cell Biol. 2, 107–117.
4. Scheidig, A.J., Burmester, C. & Goody, R.S. (1998) Use of caged
nucleotides to characterize unstable intermediates by X-ray crys-
tallography. Methods Enzymol. 291, 251–264.
5. Ahmadian,M.R.,Zor,T.,Vogt,D.,Kabsch,W.,Selinger,Z.,
Wittinghofer, A. & Scheffzek, K. (1999) Guanosine triphos-
phatase stimulation of oncogenic Ras mutants. Proc. Natl Acad.
Sci. USA 96, 7065–7070.
6. Gruen, M., Becker, C., Beste, A., Reinstein, J., Scheidig, A.J. &
Goody, R.S. (1999) 2¢Halo-ATP and -GTP analogues: rational
phasing tools for protein crystallography. Protein Sci. 8, 2524–
2528.

new titration calorimeter. Anal. Biochem. 179, 131–137.
16. Franken, S.M., Scheidig, A.J., Krengel, U., Rensland, H.,
Lautwein, A., Geyer, M., Scheffzek, K., Goody, R.S., Kalbit-
zer,H.R.,Pai,E.F.&Wittinghofer,A.(1993)Three-dimen-
sional structures and properties of a transforming and a
nontransforming glycine-12 mutant of p21
h–Ras
. Biochemistry 32,
8411–8420.
17. Schweins, T., Geyer, M., Scheffzek, K., Warshel, A., Kalbitzer,
H.R. & Wittinghofer, A. (1995) Substrate-assisted catalysis as a
mechanism for GTP hydrolysis of p21
ras
and other GTP-binding
proteins. Nat. Struct. Biol. 2, 36–44.
18. Herrmann, C., Martin, G.A. & Wittinghofer, A. (1995) Quanti-
tative analysis of the complex between p21
ras
and the Ras-binding
domain of the human Raf-1 protein kinase. J. Biol. Chem. 270,
2901–2905.
19. Wu
¨
thrich, K. (1986) NMR of Proteins and Nucleic Acids.John
Wiley &. Sons, Inc, New York.
Ó FEBS 2002 Nucleotide analogues GTPcNH
2
and GTPcF(Eur. J. Biochem. 269) 3277
20. Vasavada, K.V., Ray, B.D. & Nageswara Rao, B.D. (1984)
31

as studied by NMR. Biochemistry 36, 5045–5052.
27. Geyer, M. & Wittinghofer, A. (1997) GEFs, GAPs, GDIs and
effectors: taking a closer (3D) look at the regulation of Ras-related
GTP-binding proteins. Curr. Opin. Struct. Biol. 7, 786–792.
28. Wittinghofer, A. & Nassar, N. (1996) How Ras-related proteins
talk to their effectors. Trends Biochem. Sci. 21, 488–491.
29.Geyer,M.,Assheuer,R.,Klebe,C.,Kuhlmann,J.,Becker,J.,
Wittinghofer, A. & Kalbitzer, H.R. (1999) Conformational states
of the nuclear GTP-binding protein Ran and its complexes with
the exchange factor RCC1 and the effector protein RanBP1.
Biochemistry 38, 11250–11260.
30. Cherfils, J., Menetrey, J., Le Bras, G., Janoueix-Lerosey, I., de
Gunzburg, J., Garel, J.R. & Auzat, I. (1997) Crystal structures of
the small G protein Rap2A in complex with its substrate GTP,
with GDP and with GTPgammaS. EMBO J. 16, 5582–5591.
31. Knorre, D.G., Kurbatov, V.A. & Samukow, V.V. (1976) General
method for the synthesis of ATP c-derivatives. FEBS Lett. 70,
105–108.
32. Babkina,G.T.,Jonak,J.,Karpova,G.G.,Knorre,D.G.,Rychlik,I.
& Vladimirov, S.N. (1988) Affinity labeling at the A-site of
Escherichia coli ribosomes by a non-hydrolyzable c-amide analog
of GTP. Biochimie 70, 597–603.
33. Haley, B. & Yount, R.G. (1972) c-Fluoroadenosine triphosphate.
Synthesis, properties, and interaction with myosin and heavy
meromyosin. Biochemistry 11, 2863–2871.
34. Pfeuffer, T. & Eckstein, F. (1976) Topology of the GTP-binding
site of adenylyl cyclase from pigeon erythrocytes. FEBS Lett. 67,
354–358.
35. Klaus, W., Schlichting, I., Goody, R.S., Wittinghofer, A., Rosch, P.
& Holmes, K.C. (1986)

Kigawa, T., Ebisuzaki, T., Takio, K., Shibata, T., Yokoyama, S.,
Smith, B.O., Laue, E.D. & Cooper, J.A. (1999) Nuclear magnetic
resonance and molecular dynamics studies on the interactions of
the Ras-binding domain of Raf-1 with wild-type and mutant Ras
proteins. J. Mol. Biol. 286, 219–232.
3278 M. Stumber et al. (Eur. J. Biochem. 269) Ó FEBS 2002