J. Chem. Soc., Pe kin T ans. 2, 1999, 1143–1148 1143
1H and 31P NMR s udy o ADP and GDP spon aneous syn hesis
om 2-me hylimidazole-ac i a ed AMP and GMP nucleo ides and
ino ganic phospha e
José Pe ei a* and Ana Cade e
CEQUP/Ins i u o de Ciências Biomédicas de Abel Salaza , Uni e si y o Po o,
La go P o esso Abel Salaza , 2, 4099-003 PORTO, Po ugal
Recei ed (in Camb idge) 2nd Feb ua y 1999, Accep ed 19 h Ma ch 1999
1H and 31P NMR da a show ha adenosine 59-(2-me hylimidazol-1-ylphosphona e) (2-MeImpA) and guanosine
59-(2-me hylimidazol-1-ylphosphona e) (2-MeImpG), known as possible p ebio ic p ecu so s o polynucleo ides,
p oduce he co esponding diphosphonucleo ides in sodium phospha e solu ion a pD 7.6. Phospha e ions also
enhance he hyd olysis o hese molecules by ac i a ing a wa e molecula associa ed as a weak complex wi h
he phospho yl moie y o 2-MeImpA and 2-MeImpG. A kine ic s udy was done by quan i a i e 1H NMR
spec oscopy and mechanis ic hypo heses we e es ed by semiempi ical PM3 modelling.
In oduc ion
Imidazole-ac i a ed nucleo ides can be o med in simula ed
p ebio ic condi ions and ha e been used as p ecu so s o
in i o syn hesis o a ious kinds o oligo ibonucleo ides1–4
and oligodeoxy ibonucleo ides5 using as empla es p e-exis en
polynucleo ides. O he lines o wo k s udy he e ec i eness o
ino ganic ca alys s o empla es, mo e likely o be p esen in
p ebio ic condi ions, like me al ions6 o mon mo illoni e clays
and o he mine als.7 De ailed mechanis ic and kine ic analysis
o nucleo ide imidazolide hyd olysis and polyme isa ion is a
undamen al s ep o unde s and he ac ion o hese pa icula ly
eac i e oligonucleo ide p ecu so s. The gene al eac ion o
polyme isa ion occu s h ough a nucleophilic subs i u ion on
he phospho yl moie y o he ac i a ed nucleo ide by ano he
nucleo ide hyd oxy g oup, imidazole being he lea ing g oup.
The MgII ion has an impo an ole in he p ocess,8 u he
ac i a ing he phospho yl moie y. Depending on he condi ions
( empla es, ca alys s, pH, empe a u e) se e al kinds and
leng hs o oligonucleo ides may esul , e en dime s like NppN
(py ophospho yl b idge be ween wo nucleo ides). The ac i-
a ed phospho yl g oup is in ac sensi i e o any nucleophile,
sugges ing a me hod o he p ebio ic syn hesis o nucleo ide
polyphospha es by eac ion wi h ino ganic phospha e o py o-
phospha e in solu ion. In his wo k, he e ec o phospha e
concen a ion on 2-me hylimidazole-ac i a ed nucleo ides
was s udied in aqueous sys ems con aining sodium phospha e
and adenosine 59-(2-me hylimidazol-1-ylphosphona e) (2-
MeImpA) o guanosine 59(2-me hylimidazol-1-ylphosphona e)
(2-MeImpG) (Scheme 1).†
NMR was chosen as he quali a i e and quan i a i e
analy ical echnique in his s udy as an al e na i e o he com-
monly used HPLC because i p o ides an equally accu a e and
mo e s aigh o wa d expe imen al me hod. I also has he
ad an age o p o iding s uc u al insigh in o he eac ion
p oduc s. The pD alue o 7.6 was chosen o he expe imen s
because i is low enough o a oid eac ion o 2-MeImpN wi h
OH2 and la ge enough o limi he eac ion a e so ha NMR
spec a acquisi ion imes a e negligible. I is also inside he
p esumed pH alues o p ebio ic sea wa e .
† In he ex 2-MeImpA s ands o 2-MeImpA2 and H-2-MeImpA±,
2-MeImpG s ands o 2-MeImpG2 and H-2-MeImpG±, 2-MeImpN
s ands o 2-MeImpA and 2-MeImpG. The wo d phospha e o he
symbol Pi designa e H2PO4
2 and HPO4
22.
Resul s and discussion
Analysis and in e p e a ion o 2-MeImpG and 2-MeImpA
hyd olysis NMR spec a
Typical 1H and 31P spec a o 2-MeImpG and 2-MeImpA in
phospha e bu e a e p esen ed in Fig. 1 a e some o he eac-
ion has occu ed. The spec a aken in 0.2  TRIS bu e
ins ead o phospha e bu e a e iden ical o he spec a in Fig.
1, bu he signals assigned o ADP o GDP species a e absen .
The chemical shi s p esen ed in Fig. 1 a e cons an o all
expe imen s epo ed in his wo k wi h espec o bu e ype,
bu e concen a ion and ime and so co espond o a ull
1H and 31P NMR spec al cha ac e isa ion o 2-MeImpA and
2-MeImpG and hei hyd olysis p oduc s, unde he s anda d
condi ions used in all expe imen s: 0.02  2-2-MeImpN, 0.5 
NaCl, pD 7.6, 308 K (see Expe imen al).
The assignmen o he 2-MeImpG spec um was pe -
o med by compa ing wi h spec a o GMP, guanosine and
Scheme 1
N
N
N
N
NH2
A
2 8
N
HN
N
N
O
G
8
H2N
NN
CH3
2
45NHN
CH3
+
2-Melm H-2-Melm+
HH
H
R1
CH2
H
OH OH
OP
R2
O
O– 1′
2-MelmpA–R1 = A, R2 = 2-Melm
H-2-MelmpA±R1 = A, R2 = 2-Melm+
2-MelmpG–R1 = G, R2 = 2-Melm
H-2-MelmpG±R1 = G, R2 = 2-Melm+
2-MelmpR–R1 = –NH2, R2 = 2-Melm
H-2-MelmpR±R1 = –NH2, R2 = 2-Melm+
5′
α