Volume 15 Number 8 1987
Nucleic Acids Research
Tbe action of a water-soluble carbodlimlde on adenosine-5'-polvpbosphates
Kam-mui Eva Ng and Leslie E.Orgel
The Salk Institute for Biological Studies, PO Box 85800, San Diego, CA 92138, USA
Received December 10, 1986; Revised and Accepted March 18, 1987
ABSTRACT
When aqueous solutions of adenosine-6'-»ono-, di-, or triphosphates are
treated with a water soluble carbodiinide the sajor product is the expected
diadenosine-5'-5'-polyphosphite.
The yields of these pyrophosphates are
greatly increased in the presence
of the Mg2+ ion.
Adenosine-5'tetraphosphate behaves differently.
The major product is adenosine-5'monophosphate.
We beliere that this hydrolysis occurs via a cyclic
triHetaphosphate intermediate.
INTRODUCTION
When adenosine-5'-triphosphate is treated with dicyclohexylcarbodiimide
in anhydrous pyridine, the /J and 7 phosphate groups are partially
interchanged (1). It is believed that the intramolecular attack of the aphosphate on the activated 7-phosphate gives an ester of the triaetaphosphate
ion, which nay then open by either of two equivalent paths (Figure 1 ) .
We have used aqueous solutions of water-soluble carbodiimides as activating agents in a variety of reactions of nucleoside-5'-phosphates that involve
the formation of phosphodiester (2), phosphoramidate (3) and pyrophosphate
bonds (4). In this context, it seened interesting to study the products
formed when aqueous solutions of nucleoside-5'-oligophosphates are treated
with a carbodiimide. Here we report on a nunber of these reactions, one of
which appears to involve the elimination of three phosphate groups from
adenosine-5'-tetraphosphate via a novel cyclic trinetaphosphate intermediate.
BTPKRDffiHTiL
Materials
Adenosine-5'-mono-, di-, tri- and tetraphosphates (AMP, ADP, ATP and
ATeP, respectively) (5) were obtained as sodiua salts from Sigma Chemical
Company,
l-ethyl-3-(3-dimethylaoinopropyl)carbodiimide
(CDI) from JBL
Scientific,
and l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
metho-p-
© IRL Press Limited, Oxford, England.
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Nucleic Acids Research
toluenesulfonate (morpho CDI)
from
Aldrich
Chemical
Co.
Calf intestinal
alkaline phosphatase and venom phosphodiesterase-I (crotmlua adaoMateus) were
purchased
from
Boehringer
Mannheim
Biochemicals
and
Pharmacia
P.-L.
Biochemicals Inc., respectively.
Chroaatography and Identification of Products
Paper chromatography was performed on Whatman 3MM paper by the descending
technique.
The following systems were used:
System I: 95% ethanol, 1 H
in EDTA and brought
In order to
get
ammonium
to
pH 5.0
improved
acetate made up to 2 x 10-3 U
with glacial acetic acid (7:3).
resolution
phates, we applied a solution
of
of the various polyphos-
0.25 M EDTA N&4 to the origin
of the chromatograa in the form of
a band about 1 cm wide.
The
polyphosphate mixtures were applied to the dried band.
Systen II: n-propanol, concentrated ammonia and water (55:10:35).
System III: 0.03 U potassium phosphate (pH 7.1).
Paper electrophoresis was performed at 3000
Each UV absorbing
spot
was
eluted
from
density was read at 259 nm against the
chromatogram.
The
yields
are
volts using varsol as a coolant.
the
chromatogram and the optical
eluate
expressed
from the same area of a blank
as
the
percentage
of the total
absorbancy of all the material on the paper.
The identity of the
alkaline
phosphatase
reaction
and
products
venom
oligophosphates were converted
was
confirmed by digestion with
phosphodiesterase-I.
to
9
adenosine
The
adenosine-5'-
on exhaustive degradation with
? ff
A-O-P-O-P-«-P-»-
6. 6. 4.
DICYCLOHEXYLCARBOOIIMIDe
A-o<j>o'»
\
WATER
o
•
o
9
9
A-O-P-O-p'-B-P-O" + A-O-P-O-P-*
6. i- 6.
6. 6-
Figur a 1: The mechanism proposed by Webb to explain the interchange of the
and 7 phosphates of ATP that occurs in the presence of a carbodiimide (1).
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Nucleic Acids Research
alkaline phosphatase.
Less complete
that co-chromatographed
with
the
degradation
series
of
gave a series of compounds
nucleoside 5'-oligophosphates
shorter than the starting material.
The
diadenosine-5'-5'-polyphosphates
phosphatase, showing
that
they
do
esterified oligophosphate groups.
garo approximately equal
not
were
contain
unaffected
by
alkaline
phosphomonoester or mono-
Short digestions with phosphodiesterase-I
amounts
of
adenosine-5'-phosphate
and a material
which had the same nobility
as
the adenosine polyphosphate with a phosphate
chain one shorter than that
of
the
degradation with
phosphodiesterase-I
nobilities of shorter
starting
gave
material (8). More extensive
a
series
adenosine-5'-oligophosphates
of
products with the
and, finally, adenosine-
5'-monophosphate.
Ensye Degradations
a) Calf intestinal alkaline phosphatase.
An aliquot of the compound con-
taining approximately 1 /(mole of adenosine was
incubated at 37°0 with 12U of
Table 1.
Chromatographic and electrophoretic nobilities.
The Rf and E m values of the various adenosine-5'polyphosphates and adenosine-5',5'-polyphosphates are
given relative to adenosine-5'-phosphate.
Ef in systen
Rn in system
n
m
1.00
1.00
0.57
0.00
1.00
1.17
0.34
0.89
0.23
0.79
0.76
1.26
1.01
1.14
0.69
ADP-Borpio Ure»
0.82
1.25
0.79
P&A
P7A
ApaA
AP3*
AP4A
ApsA
Ap«A
0.14
0.09
0.71
Aj*A
0.04
0.62
Coapound
AMP
ADP
ATP
ATeP
ADP-Ore»
I
0.06
0.63
1.14
0.34
0.96
0.21
0.82
0.13
0.80
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Nucleic Acids Research
Table 2.
Products froa the reaction of CDI with adenosine-5'-polyphosph*tes.
For reaction conditions sas text.
NucleoTioe Mg2^
side
AMP
2h
4k
ADP
+
+_
6h
+
24h
+
2h
+
ADP-
AMP Urea ADP Ap-2A A P 3 A ATP ATeP A P 4 A A P 5 A ApeA a Ap8J
16.1
80.6
13.4
77.5
80 .0
18 .7
84 .7
19 .4
86 .6
22 .5
12.3
75.9
87 .6
24 .1
20.0
81.3
9.8
4h
+
6h
+
11.8
12.7
24h
+
13.9
ATP
2h
+
4h
+
-
24h
ATeP
2h
3.5
21.1
2.5
2.8
21.9
36. 9
38.2
3.9
5.5
20. 3 17. 5
39.1
5.3
17. 6 17. 0
39.9
5.0
15. 3 16. 1
39.9
4.8
15. 3 14. 6
+
+
+
6h
+
+
a) contained snail amount of unresolred ApsA
b) contained small amount of unresolTed Ap7A
3576
52.5
27.2
64.7
2fl.O
65.3
33.2
66.9
35.4
5 .2
2 .2
7 .9
3 .4
9 .9
4 .0
11 .6
5 .6
2.6
22.5
1.6
21.7
+
4h
24h
33.1
52.2
12.3
39.7
7.5
31.1
2.1
22.0
4.0
45. 9
2. 3
41. 1
1. 4
38. 7
1.8
6h
9 2
8 8
15 7
16 1
17 3
19 0
19 .4
23 1
2.9
7.1
4.7
6.7
6.5
6.2
9.5
6.1
89
25
90
27
90
30
90
32
13.0
7 .9
17.1
7 .4
16 .6
8 .3
15.6
10.7
6
8
5
9
5
9
5
3
81. 4
12. 2
82. 9
13. 6
83. 4
14. 4
84. 4
14. 8
Nucleic Acids Research
fTEMUNAL)
^S^
/
ATJ^\
I ATTACK ) ^ ^ . . _ /
^
fTEMDMl-l
H
^ \ .
^/mn..rn.^-n^
\ ^ l ATTACK J
^
* I ATTACK
J
A-O(P-O]
6.-
AM
~-$°t\r
f-O-
b^b
A-Ofl-O)—P-O-
A-o{I-0>—f-O-A
IV (b-d)
V (b-d)
Figure 2: A scheme that accounts for all of the products formed by the
action of a water-soluble carbodiimide on AMP, ADP, ATP and ATeP. a) n = 0;
b) n = 1; c) n = 2; d) n = 3.
the enzyme in 20 pi of 0.05 11 Tris
buffer, pH 9.0.
After 3 hr. incubation,
an aliquot was withdrawn and examined by paper chromatography in System I.
b) Venom phosphodiesterase-I. Approximately
incubated at 37°C with
phosphodiesterase-I.
20 fil of 0.05 M Tris
1 /(mole
of the compound was
buffer,
pH 0.0, and 0.2U of
Aliquot3 were removed after 5 sin. in the case of ApgA,
15 min. in the case of ApsA or Ap&A, 30 min. in the case of Ap3A or Ap4A, and
50 min. in the case of Ap2A, and chronatographed directly in System I.
Preparation of samples
The reactions were
carried
out in Eppendorf
tubes (1.5 aL).
Aqueous
solution containing ATeP, ATP, ADP or AMP (50 mil), Hepes (2 11), CDI (2.5 II)
were thoroughly nixed with a vortex mixer and incubated at 37°C, pH 0.5. The
total reaction Tolume was 100 pi.
In another
concentrations of reagents were used
set of experiments the same
and, in addition, 125 DM, 100 mM, 75 mM
or 50 mil MgCla for ATeP, ATP, ADP or AMP, respectively.
The pB was adjusted so that
temperature.
The pH of each
end of the experiment using
each
mixture
mixture
was at pH 0.5 at the reaction
was measured at the beginning and the
a Microelectrodes, Inc. MI-410 micro combination
pH probe and a Beckman Model 4500 digital
pH neter.
The pB was always found
to remain unchanged throughout an experiment.
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Nucleic Acids Research
RESULTS AMD DISCOS3I0N
The major products of the action of
and ATP are the
ApgA respectively (Table 2).
ADP and of ADP to AMP, and
Ap5A from ATP.
System I.
CDI on aqueous solutions of AMP, ADP
anticipated diadenosine-6'-5'-polyphosphates, Ap2A, Ap4A and
In
Side reactions lead to the hydrolysis of ATP to
to
addition,
the
synthesis
ADP
yielda
The major product from the
ATeP gives some ApgA, ApsA and
ATeP can be increased to
ATP.
as much
of ApjA from ADP, and Ap4A or
a
product
reaction
with an Ef of 0.76 in
of ATeP is AMP.
as
90%
if
the concentration of ATeP is
decreased to 0.01 M and the concentration of CDI is 1.0 M.
CDI,
In addition
The yield of AMP obtained directly from
the nucleoside-5'-polyphosphites are
In the absence of
not significantly hydrolysed in 24
hra. under the same conditions.
The compound with an Rf of
unaffected
by
alkaline
0.76
formed
phosphatase
phosphodiesterase-I to yield AMP.
It
by
out
is
degraded
by
venom
seems plausible, therefore, that it is
an adduct of CDI with the P phosphate of ADP.
adduct is formed, we carried
the action of CDI on ADP is
but
To test the hypothesis that an
a reaction using a different water-soluble
VI
CARBOOIIMICe
NHR'
P-O-C
A-
6
pm»O"
6.
.0
O-
AMP + TRItETAPKOSPHATE
Figure 3: A proposed mechanism for the formation
presence of a water-soluble carbodiimide.
3578
of
AMP from ATeP in the
Nucleic Acids Research
carbodiimide, norpho CDI,
in
place
of
CDI.
Wo
obtained a product with
properties very similar to that previously obtained with CDI, but which could
be resolved from it by
1).
paper
chromatography or paper electrophoresis (Table
Analogy with the work of
Khorana (7) suggests, but does not prore, that
these conpounds are N-phosphorylureas (I), formed by the rearrangement of the
isoureas
that
result
from
the
attack
of
ADP
on
the
water-soluble
carbodiiaides.
o
o R' o
II
II I
II
II
A-O-P-O-P-N-C-NHR
6- 6.
I
The presence of divalent metal ions
is known to facilitate reactions in-
volving the nucleophilic attack of one phosphate anion on the activated derivative of another
(8).
It
catalyses the synthesis of
is
not
surprising,
therefore, that the Mg2+
diadenosine-5'-5'-pyrophosphates and leads to 80-
Q0% yields in most cases (Table 2 ) .
All of the observed products can
first product of the reaction is
the terminal phosphate
on
the
be
accounted for if we assume that the
always
the isourea formed by the attack of
carbodiiaide
(Figure 2).
The formation of
pyrophosphates containing twice the number of phosphate groups present in the
starting naterial Bust
activated substrate.
involve
attack
of
Hydrolytic removal
by water attack on the penultimate
phate groups, which must be
sore
extensive,
substrate with a
The
molecule
of
its
first
for attack on the penultimate
is
condensation
Attack on the terminal phoswould not be detected since it
Precedent
phosphate of a nudeoside-S'-polyphosphate
(0).
unactivated substrate on the
a single phosphate group occurs
phosphate.
regenerates the starting naterial.
and his co-workers
an
of
found
of
a
in the work of Hoffatt
molecule of the initial
hydrolysis
product
leads to the
synthesis of Ap3A from ADP, etc.
The simplest explanation of
the
direct
and efficient production of AMP
from ATeP, is the participation of a cyclic covalent intermediate V H (Figure
3). This intermediate is analogous to that involved is the interchange of the
P and 7 phosphates of ATP
(0)
(Figure
1).
It may reopen to give starting
material, or eliminate inorganic trimetaphosphate
to
give AMP.
The conden-
sation of AMP with ATeP then leads to an appreciable yield of ApsA.
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Nucleic Acids Research
ACKNOILBDCBMEMTS
This work v a s supported by grant #01113435 fron t h e N a t i o n a l I n s t i t u t e s of
Health. We thank S y l v i a B a i l e y f o r manuscript p r e p a r a t i o n .
1. lebb.M.R. (1980) Biochea. 18, 4744-4748.
2. Inoue.T. and Orgel.L.B. (1982) J. Mol. Biol. 162, 201-217.
3. Chu,B.C.F., fahl.G.M. and Orgel,L.E. (1983) Nucleic Acids Research 11,
0513-6529.
4. Chu.B.C.F. and Orgel.L.E. (1984) Biochia. Biophjs. Act* 782, 103-105.
5. Abbreviations: AMP, ADP, ATP, and ATeP, psA, p7A refer to the homologous
series of adenosine-S'-nono-, di-, tri-, tetra-, penta-, and
heptaphosphates. Ap2A, Ap3A, Ap4A, ApsA, ApQA and ApgA refer to the
hoaologous series of a,«-di(adenosine-5')polyphosphates; e.g., ApgA is
pl,p8-di(adenosine-5')octaphosphate.
ADP-Urea and ADP-morpho Urea
refer to the adenosine-5'-diphosphoryl-(N-ethyl,N'-dinethylandnopropyl)-urea and adenosine-5'-diphosphoryl-[N-(2-morpholinoethyl),N'cyclohexyl]-urea.
6. Lohrman.B. (1875) J. Mol. Brol. 6, 237-262.
7. Khorana,H.,G., Turner,A.P. and Viasolyi,J.P. (1881) J. Amer. Chea. Soc.
83, 686-688.
8 . Burton,K. and Krebs.H.A. (1863) Biochea. J. 6 4 , 8 4 - 1 0 7 .
8 . a) Yerheyden,D.L.ll., f e h r i l , f . B . and U o f f a t t . J . G .
(1865) J. Amer. Chea.
Soc. 8 7 , 2257-2265.
b) f e h r i l . W . E . and M o f f a t t , J . G . (1865) J. Aaer. Chea. Soc. 8 7 , 3760-3766.
3580