All of these methods produce nucleotide phosphorothioates as racemic mixtures, although the diastereoiso-
mers can generally be resolved by reversed phase HPLC (the Spdiastereoisomer always elutes first) and
characterized by^31 P NMR (the signal for the -phosphate of the Spdiastereoisomer is downfield).108,112
Nucleotide thioesters have become prime tools for the investigation of the stereochemistry of enzyme-
catalysed phosphoryl transfer processes.^112 For example, the (Sp) isomer of adenosine 5-O-(1-thio)triphos-
phate, ATPS, is readily made from AMPS by the combined action of adenylate kinase and pyruvate kinase
(both enzymes can be immobilized on a polymer support for large scale syntheses) and by use of phos-
phoenol pyruvate with a little ATP to start the cycle. This synthesis illustrates the stereospecificity of
adenylate kinase. The^31 P NMR of this product has been used to identify the (Rp) and (Sp) diastereoisomers
of dATPS, which have been synthesised and separated by HPLC. Such species have been employed inter
aliaas substrates for DNA and RNA polymerases, which only incorporate nucleotide thiotriphosphates of
the Sp configuration, to show that polymerases, as well as T4 RNA ligase and adenylate cyclase, operate
on adenine nucleotides with inversion of configuration at P.^113 For such purposes, ATP has been made
with incorporation of either^17 O or^18 O in just about every possible position in the three phosphate residues.^114
The more useful species for nucleic acid chemistry are the -phosphate substituted nucleotides. These can be
made either by ab initiosynthesis or the stereochemically controlled replacement of sulfur of an -thio-
phosphateresidue by isotopic oxygen. This transformation is best carried out by controlled bromine oxi-
dation in^17 O- or^18 O-enriched water (Figure 3.68). While this reaction proceeds with inversion of
configuration, similar oxidations with N-bromosuccinimide or cyanogen bromide have been found to be less
stereoselective. An alternative procedure, although less widely applied, has used [^18 O]-styrene oxide, when
the substitution of sulfur by oxygen proceeds with exclusive retention of stereochemistry at phosphorus.
The P^1 ,P^2 -diesters of pyrophosphoric acid are most often made by coupling together two phosphate
monoesters using DCC, by a morpholidate procedure or by diphenyl phosphorochloridate.115–117A clas-
sical example is Khorana’s synthesis of co-enzyme A.^118 The same methods have worked well for syntheses
of Ap 4 A and its analogues, where the use of an excess of activated AMP and limiting pyrophosphate (or one
of its analogues) gives acceptable yields of P^1 ,P^2 -dinucleosidyl tetraphosphate or analogue (Figure 3.69).
Making the P^1 ,P^2 -dialkyl triphosphates of the mRNA ‘cap’ structures has called for more sophisticated
coupling procedures.^119 This is partly on account of the lability of the glycosylic bond in the 7-MeGuo
residues and partly because of the unsymmetrical character of the diester (Figure 3.70). In general, the
major problem encountered in the syntheses of all these species has arisen during purification as there
appears to be no good alternative to ion-exchange chromatography, although high-performance reverse
phase silica chromatography has some uses.
Nucleosides and Nucleotides 115
OAcOPO B'P O
O
POOOOO
R'OHO RO B
P
OO
XP
O
P
O
OOO OOAcOO B'CO
P
OOR'OAcOHO B'R'ON 3O Thy
P
OO
SP
O
P
S
OOO OR' = OAc, H, F
B' = Adebz, Cytbz, Guaib, Ura or Thy(i) (ii)R = OH, H, F
B = Ade, Cyt, Gua, Thy, Ura
X = O,S, BH 3(iii)Figure 3.67 Syntheses of nucleoside triphosphates and analogues using phosphorus(III) chemistry. Inset AZT-a,
g-phosphorodithioate formed through ring opening of cyclic thiotriphosphate with Li 2 S in DMF.
Reagents: (i) salicyl chlorophosphite in dioxan/DMF; (ii) tetrakis (tributylammonium) pyrophosphate
in DMF; and (iii) XO: iodine in aq pyridine, then NH 4 OH; XS: S 8 , then NH 4 OH; XBH 3 :
borane-i-Pr 2 EtN complex, then NH 4 OH