A GENERAL SYNTHESIS O F NUCLEOSIDE-5' TRIPHOSPHATES1

J . G. MOFFATT
Syntex Institute for Afolecular Biology, Palo Alto, California

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Received August 15, 1963

ABSTRACT
The reaction between ribo- and deoxyribo-nucleoside-5' phosphoromorpholidates and
tributylammonium pyrophosphate in molecular sieve dried dimethyl sulphoxide has been
found to give the nucleoside-5' triphosphates in 81-84% yields. A convenient work-up of
the triphosphates following ion exchange chromatography permits isolation of the triphosphates
in overall yields of 73-80% as the pure sodium salts. -4 number of examples of the reaction
are given.

Progress in biochemical research during recent years has clearly pointed out the unique
importance of ribo- and deoxyribo-nucleoside-5' triphosphates in biological systems.
Thus, in addition to the well-ltnown role of adenosine-5' triphosphate (ATP) as the primary energy source in many systems, the nucleoside triphosphates also serve as direct
precursors of both ribonucleic (1) and deoxyribonucleic ( 2 ) acids, and of nucleotide
coenzymes (3). Certain triphosphates also act as rather poorly understood specific cofactors for certain biological transformatioils (4). This multiplicity of function of nucleoside
triphosphates has led to a demand for efficient synthetic routes to the compounds, and a
considerable effort has been made in this directlon (5). The general method which has
been most widely used to date consists of the reaction of a nucleoside-5' phosphate with a
large excess of inorganic orthophosphate in the presence of dicyclohexylcarbodiimide (6).
This method was much improved when it was realized t h a t the reaction could be conducted
in anhydrous pyridine if the tributylamine salts of the various acidic components were
used (7). This modification allowed the reaction to be run for the first time in a homogeneous system and also led to the selective formation of the nucleoside triphosphates in
considerably higher yield than the other mixed products. In order to achieve a satisfactory
yield, however, it was necessary to use a t least 10 molar equivalents of orthophosphate.
The presence of the resulting large excess of mixed inorganic polyphosphates formed
during the reaction necessitated selective adsorption of the nucleotides on carbon followed
by elution with ethanolic ammonia prior to ion exchange chromatography. Such a step
frequently leads to considerable losses of the nucleotidic material and usually the final
isolated yields are far below those present in the crude reaction. In spite of this, the
simplicity of the method has made this the most widely used route to nucleoside triphosphates (8).
T h e development of a simple general method for the conversion of nucleoside-5'
phosphates into activated but insolable phosphoramidate (9), and in particular phosphoronlorpholidate (lo), derivatives (I) has opened the way t o syntheses, in high 1-ield, of
nucleoside-5' diphosphates (111, R' = FI) and nucleotide coenzynles (111, R' = sugars,
etc.) by their reaction with orthophosphate (11, R' = H) or various phosphate monoesters (11, R' = sugar, etc.) in anhydrous solvents (10, 11, 12).
In an earlier paper ( l o ) , the attempted extension of the above reactions to the direct
synthesis of a nucleotide-5' triphosphate through the reaction of adenosine-5' phosphoronlorpholidate (IV, R = OH, R' = adenine) with tributylainmonium pyrophosphate in
'Contribution No. 3 from the Syntex Institute for Molecular Biology, Palo Alto, California.
I

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1964

anhydrous pyridine was described. This reaction led rather rapidly to a moderate yield
(57y0) of adenosine-5' triphosphate (V, R = OH, R' = adenine) within 2 hours but on
prolonged reaction this initial product disappeared and was converted into adenosine-5'
diphosphate, which was present in 76y0yield after 24 hours. Further studies on this reaction (13) have shown it to be quite general, the rate of disappearance of the initially

formed triphosphate being a function of the particular nucleotide under consideration

(14).* I t is, moreover, possible to conduct the reaction using only 3-4 molar equivalents
of pyrosphosphate without appreciable formation of P1,P4-dinucleoside-5' tetraphosphates
(13). Since the excess pyrophosphate is not chemically activated, and hence remains
substantiaIly unchanged, it is possible to separate the resulting nucleotides by ion exchange chromatography without the necessity of charcoal absorption. Several other
workers (15) have also studied the reaction of nucleoside-5' phosphoramidates with
pyrophosphate in a variety of solvents but only in a recent study by Ikehara and Ohtsuka
( i 5 ( d ) ) has any mention been made of the disappearance of the initial products, and the
filial yields have not been sufficiently good t o make this route superior to the carbodiimide
reaction. Only in the work of Tanaka et al. (15(b)) was pure pyrophosphate free of large
amounts of orthophosphate used, thus allowing a critical examination of the desired
reaction.
I t was of considerable importance to devise reaction conditions such that degradation
of the initially formed triphosphates did not occur and this product could be isolated in
high yield. As a result of another study in this laboratory (141, it was observed that while
tributylaminonium adenosine-5' triphosphate was rapidly converted into a complex
mixture of products in pyridine (10) it was virtually completely stable in a
number of other anhydrous organic solvents. One such agent is dimethyl sulphoxide
which offers particular interest in view of its high solvent power for nucleotides.
Accordingly the reaction of deoxyadenosine-5' phosphoromorpholidate (IV, R = H, R' =
adenine) with tributylammoniunl pyrophosphate was studied in dimethyl sulphoxide.
Initially the two components were separately rendered anhydrous by coevaporation
with pyridine and then mixed in distilled dimethyl sulphoxide. The reaction proceeded
*Veiheyden and Moffatt will describe shortly a detailed .ctudy of the interesting decomposition of the nucleoside
polyphosphates in certain anlzydrous organic solvents.

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MOFFXTT: NUCLEOSIDE-5' TRIPHOSPHATES

quite rapidly, the morpholidate having almost completely disappeared after 10 hours,
and the product distribution remaining unchanged after 24 hours. The products were
separated by ion exchange chromatography on a DEAE cellulose column in the bicarbonate form using a linear gradient of triethylammonium bicarbonate as eluant." Under
these conditions, the major product was indeed deoxyadenosine-5' triphosphate (54%).
Only small amounts (2% and 1Oy0 respectively) of deoxyadenosine-5' phosphoromorpholidate and deoxyadenosine-5' diphosphate were present but deoxyadenosine-5'
monophosphate comprised 34% of the total nucleotides. This picture was surprisingly
reproducible under a variety of reaction conditions including further coevaporation of the
final reaction mixture with pyridine and dropwise addition of the morpholidate. When,
however, the dimethyl sulphoxide was dried by standing for several days with a moiecular
sieve? and then used in a similar reaction, the yield of deoxyadenosine-5' triphosphate
rose to 82% with only 6%, 7%, and 5% respectively of the unreacted morpholidate,
monophosphate, and diphosphate. I t appears, therefore, that, as was found earlier ( 1 0 , l l )
in experiments leading to the synthesis of nucleotide coenzymes in pyridine, it is essential
to maintain conditions as anhq-drous as possible to avoid simple hydrolysis of the morpholidate. I t is interesting to note that when molecular sieve dried dimethyl sulphoxide
was used the reaction becomes much slower, usually 48-72 hours being necessary for
disappearance of nearly all the morpholidate.
Isolation of the product in a water-soluble, non-hygroscopic, solid form is readily
accomplished by removal of excess triethylammonium bicarbonate by low temperature
evaporation, and adding an excess of sodium iodide in acetone to a concentrated solution
of the resulting triethylammonium salt of the nucleotide in methanol. Recoveries of the
resulting sodium salt are generally excellent and the products as directly obtained are
suitable for use in biochemical studies.
This general procedure has been used for the synthesis of all the normal deoxynucleoside-5' triphosphates and also several ribonucleoside-5' triphosphates. The results are
summarized in Table I and it can be seen that in all cases the yield of pure isolated sodium
TABLE I

Product distribution of the various nucleoside polyphosphates

yo yields based

on starting material

Kucleoside

Morpholidate

MonoPo4

DiP04

Trip04

Isolated Na
salt of Trip04

Deosyadenosine
Deoxycytidine
Deoxyguanosine
Thymidine
Cytidine
6-Azauridine

6
6
3

7
5
7

4
5
9

5
7
6
9
7
5

82
82
82
81
84
82

73
80
77
75
80
77

4
7

salt fell in the range 70-80% thus being much higher than those by any previous method.
Several triphosphates have also been prepared from P32-labe1edribo- and deoxyribonucleotides in connection with another problem which will be reported on a t a later
date (16).
* I t i s our expeiience that i t i s zery dificult to isolate nucleoside polyphosphates (particularly the tri- or higher
phosphates) jronz columns i n the chloride form without minor decomposition to the lower homologues. T h i s does
not appear to be a problem with the bicarbonate columns.
jType 10X obtained through the courtesy of the Linde Co., Los Angeles. More recently we have been informed
by this colizpany that type 4 A might be tha agent of choice for drying dimethyl sulphoxide.

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I t is interesting to note the remarkable consistency of product distribution for the

various reactions summarized in Table I. The small amount of diphosphate which invariably appears seems not t o be due to the presence of orthophosphate in the reagent
grade sodium pyrophosphate used in these experiments. Indeed measurement of the
orthophosphate content by the method of Lowry and Lopez (17) shows that less than
O.lyoof the total phosphorus is orthophosphate. I t is particularly to be emphasized that
in no case is there any contamination of the triphosphate peak by inorganic phosphates
even without prior necessity of charcoal adsorption and elution. The combination of
simplicity, high yield, and purity of the products clearly makes the procedure of unique
value in the synthesis of nucleoside triphosphates. The efficient syntheses of nucleoside-5'
diphosphates previously reported using pyridine as the solvent (10) perhaps make the
use of dimethyl sulphoxide unnecessary in these syntheses but an extension of the previous
method to the direct synthesis of ~lucleoside-5' tetraphosphates using tripolyphosphate
will be described a t a later date.
EXPERIMENTAL
Paper chromatography was conducted by the descending technique on Schleicher and Schuell KO. 589
Orange Ribbon paper using the following solvent systems: solvent I , isobutyric acid : 1 S ammonium
hydroxide : 0.1 Ad tetrasodium ethylenediamine tetraacetic acid (60:100:1.6); solvent 11, n-propyl alcohol :
ammonium hydroxide : water (6:3:1). Rf's of all pertinent compounds in solvent I are given in Table I1
TABLE I1
Compound

solvent I

Compound
I

Deoxyadenosine-5' phosphate
Ueoxyadenosine-5' diphosphate
Deoxyadenosine-5' triphosphate
Deoxycytidine-5' phosphate
Deoxyc);tidine-5' diphosphate
Deoxycytidine-5' triphosphate
Deoxyguanosine-5' phosphate
Deoxyguanosine-5' diphosphate
Deoxyguanosine-5' triphosphate

Thymidine-5' phosphate
Thymidine-5' diphosphate
Thymidine-5' triphosphate
Cytidine-5' phosphate
Cytidine-6' diphosphate
Cytidine-5' triphosphate
6-Xzauridine-5' phosphate
6-Azauridiile-5' diphosphate
6-Azauridine-5' triphosphate

Rf in
solvent I1
0
0
0
0
0
0
0
0
0

44
30
21
43
29
21
2.2
16
12

..

with the exception of t h e morpholidates which decompose in this system. I n all cases the disappearance of
the morpholidate may be conveiliently followed using solvent I1 in which they move far ahead of the
corresponding monophosphates. Phosphorus-containing compounds were localized on paper chromatograms
using the Hanes and Isherwood spray (18) followed b y ultraviolet irradiation (19). Total phosphorus
determinations were done by the method of King (20) and ultraviolet spectra were recorded on a Cary
Model 15 spectrophotometer. Elemental analyses (other than for phosphorus) were obtained by Alidwest
Microlab Inc., Indianapolis, Indiana. All evaporations were done a t approximately 1 mm pressure using a
rotary-type eraporator in which the condensing bulb was cooled with circulating aqueous glycol a t -10'
t o -20" C.
material \+-as prepared aild
Deoxyadenosinc-5' phosphovowtorpholidate ( I V , R' = adenine, R = H).-This
isolated in 91y0 yield as its 4-inorpholine N,K'-dicyclohexylcarboxamidiniumsalt by the general procedure
previously reported (10). As directly obtained the chromatographically pure material (Rf 0.54 in solvent 11)
was a tetrahydrate (equivalent weight 776 by spectra) b u t after drying in 80" i n vacuo the dihydrate was
obtained. Calc. for C31H52nT907P.2HzO: C, 51.00; H , 7.73; N, 17.28. Found: C, 50.33; H, 8.23; S,17.36.
Deoxyguanosine-5' phosplzorotnorplzolidate ( I V , R' = guanine, R = H).-Isolated
as the tetrahydrate of
salt in 95% yield by the usual procedure (10). Rf0.35
its 4-morpholine N,Nf-dicyclohexylcarboxamidinium
in solvent 11. Drying i n vacuo a t 80' gave the monohydrate. Calc. for CjlH52N908P.H?O: C, 51.18; H, 7.48;
N, 17.33. Found: C, 52.02; H, 8.23; n', 17.27.
6-Azauridine-6' phospl~oromovpholidate( I V , R' = 6-a~auracil,R = OH).-This material was obtained in
the usual way (10) from 6-azauridine-5' phosphate* and isolated ill 89% yield as its chro~natographicall~
pure
*Prepared by phosphorylation of 2',3'-0-isopropylidine 6-azau~idineby the cyanoetlzyl phosplzate nzethod
(see ref. (M)). Details will be published slzortly.

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MOFFATT: TUCLEOSIDE-5' TRIPHOSPHATES

603

4-morpholine iY,Nt-dicyclohexylcarboxamidinium
salt. Rf0.36 in solvent 11. Calc. for C e 9 H s o N 7 0 i o P ~ H 2 0 :
C, 49.38; H , 7.43; N, 13.90. Found: C, 49.04; H, 7.86; N, 14.08.
ATz~cleoside-5' triphosphates. General procedure.-Reagent
grade tetrasodium pyrophosphate.10H20
(1 mmole, 446 mg) is dissolved in water and passed through a column containing Domex 50-\T' resin in the
pyridine form (15 ml). The resin is then washed with water (30ml) and the total effluent evaporated in vacuo
to a volume of approximately 10 ml. Pyridine (30 ml) is then added follo\ved by tri n-butylainine (1.0 inl,
4.2 mmoles). T h e resulting homogeneous solution is evaporated to a syrup and tendered anhydrous by four
evaporations with 10-ml portions of pyridine (dried by distillation from, and stored over calciurn hydride).
The residual pyridine is then removed b y two evaporations with 5-ml portions of calcium hydride dried
benzene. Separately the 4-morpholine K,N1-dicyclohexylcarboxamidiniumsalt of the nucleoside-5' phosphoromorpholidate (0.25 mmoles) is dried by two evaporations with 5-ml portions of pyridine followed by
t ~ v oevaporations of the residue with benzene (5 ml each). The pyrophosphate is then transferred onto the
dried morpholidate with the aid of four 1.0-ml portions of dimethyl sulphoxide* which had been dried by
distillation and storage for a t least several days, over Linde NIolecular Sieve Type 10X. The resulting clear
solution is kept a t room temperature for 2 t o 4 days, the disappearance of the morpholidate being readily
followed by chromatography in solvent 11. \47ater (30 ml) is then added and the solution is directly applied
to a 2x35-cm column of DEAE celluloset in the bicarbonate form. The column is then washed with water
until the optical density of the effluent falls t o zero and the components are eluted using a linear gradient of
triethylainlnoniuin bicarbonate. I n each case the mixing vessel initially contains 1500 ml of water and the
reservoir contains 1500 ml of 0.35-0.45 M triethylammonium bicarbonate depending upon the individual
nucleotides being separated. I n the cases of deoxyadenosine, thymidine, deoxycytidine, and cytidine-5'
triphosphate, 0.35 M salt is used while with deoxyguanosine and azauridine-5' triphosphates 0.40 Jiand
0.45 AT respectively are necessary. In each case four clearly separated ultraviolet-absorbing peaks are observed and the recovery of optical density is quantitative. The product distributions are shown in Table I.
The pooled triphosphate peak is evaporated to dryness with a bath temperature of 30-35", and all residual
triethylaminoilium bicarbonate removed by four evaporations with 25-1111 portions of methanol. The final
residue is dissolved in approximately 5 ml of methanol and a 1 M solution of sodium iodide in acetone (6
equivalents relative to the nucleoside by spectra) is added followed by 75 ml of acetone. The precipitate is
collected by centrifugation, washed three times with 30-ml portions of acetone, and dried overnight i n vacuo
over phosphorus pentoxide a t room temperature. In each case the product is a white, freely water-soluble
and non-hygroscopic powder which is chroinatographically homogeneous in solvents I and 11.
Deoxyadenosine-6' triphospJz.ate (V, R' = adenine, R = H).-The final isolated yield of the tetrasodium
~ ~ . ~H,H2.87;
Z OK
: , 11.06. Ratio of P:deoxysalt hexahydrate was 73y0. Calc. for C ~ O H ~ Z Y S O ~ ~ P ~C,S18.97;
adenosine,$3.0. Found: C, 19.06; H , 3.12; N, 10.91; P/dAdenosine, 3.12.
Deoxycytidine-6' triphosphate (V, R' = cytosine, R = H).-The tetrasodium salt monohydrate was isolated
in 80% yield. Calc. for CsHlzN30laP3Na4.HzO: N, 7.33; P/dCytidine, 3.0 Found: N, 7.29; P/dCytidine, 2.93.
Deoxyguanosine-5' triphosphate ( V ,R' = guanine, R = H).-The tetrasodium salt trihydrate was isolated
in 7757" yield. Calc. for C10H12K5013P3Na4.3H20:nT, 10.79; PldGuanosine, 3.0.Found: K, 10.75; P/dGuanosine, 2.90.
Thymidzne-5' triphosphate ( V , R' = tltynzine, R = H).-The tetrasodium salt dihydrate \\as isolated in
75% yield. Calc. for C1oH13X2O14P3Ka4.2Hz0:K, 4.62; P/Thymidine, 3.00. Found: N , 4.65; PIThymidine,
3.15.
Cytidine-5' t~iphosphate( V , R' = cytosine, R = OH).-The anhydrous tetrasodiurn salt was isolated in
80% yield. Calc. for C9Hr2S3014P3Na4: N, 7.36; P/Cytidine, 3.00. Found: N, 7.30; P/Cytidine, 2.94.
6-Azau~idine-6'triphosphate ( V , R' = 6-aeauracil, R = OH).-Isolated as the trisodium salt dihydrate
in 77y0 yield. Calc. for C8HloN30lsP3Nac.2H20: N, 7.11; P/Azauridine, 3.0. Found: N, 6.98; P/Azauracil,
3.12.
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tBrown Co., Berlin, 1V.H.
SNucleoside content is determined spectrally.

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