Hydrolysis of Amino Acid Nucleoside Phosphoramidates
FULL PAPER
conformations of the base are equal in free energy. This pro-
tonation can favor the possibility of forming a hydrogen
bond between the N3-H and the phosphate group. Hydro-
gen bonding between the base and the catalytic water mole-
between the syn and anti conformations of the nucleobase.
The JH1’-C4 coupling constant of approximately 2 Hz for
3
Asp-dAMP (1) was observed at both pD values, indicating a
predominantly anti orientation (Figure S14b in the Support-
ing Information).[38] The anti conformation of the nucleo-
base was further supported by the observed 1H–1H NOE
cross peaks (Figure S14c in the Supporting Information).
However, in case of Asp-1-deaza-dAMP (21), a significant
conformational difference in the nucleobase orientation was
observed in acidic and basic solutions. In basic solutions
cule, as shown in PO_ts2_base (Figure 5), reduces the barri-
À1
À
er for P O cleavage by 6.6 kcalmol . Although, there is
also an alternative possibility with a slightly higher energy
in which the protonated N3 of the nucleobase can directly
be involved in hydrogen bonding with the 5’-O of the nu-
cleoside part, thereby effectively delivering a proton to the
leaving nucleoside. Together these may explain the in-
3
(pD 8), the JH1’-C4 coupling constant was approximately
À
creased rate of the P O bond hydrolysis for Asp-1-deaza-
dAMP compared to Asp-dAMP, Asp-7-deaza-dAMP, and
Asp-3-deaza-dAMP.
2.0 Hz, indicating a predominantly anti conformation of the
nucleobase (Figure 6a), which is further supported by the
observed NOE cross peaks. The H8 proton showed cross
peaks with the sugar and aspartyl protons, whereas the H2
proton did not show any correlation (Figure 6b, left). More-
over, in the HOESY spectrum, the H8 proton showed a cor-
relation with phosphorus, whereas H2 did not (Figure 6c,
left). These data clearly indicate that the nucleobase orien-
tation in 21 in basic solution is in the anti domain. On the
Conformational study by NMR spectroscopy: To investigate
the role of conformation in determining the kinetics and
mechanism of P O bond hydrolysis, as suggested by molec-
ular modeling, a comparative conformational study in solu-
tion for Asp NPs containing adenine and deazaadenines was
performed in acidic and basic solutions (pD 5 and 8). The
deoxyribose sugar ring in all of the conjugates adopts a typi-
cal S-type (South, 2’-endo) puckering in D2O, irrespective of
the pD of the solution, as indicated by the appearance of a
triplet H1’ peak (Figure S12A in the Supporting Informa-
À
3
other hand, in acidic condition (pD 5), the JH1’-C4 coupling
constant was not possible to determine because the C4
carbon peak became too broad (Figure 6a), possibly indicat-
ing conformational dynamics across the glycosyl bond. How-
ever, from the ROESY and HOESY spectra, the existence
of some molecules in the syn conformation was identified
due to the presence of H2 proton cross peaks in addition to
those of H8 (Figure 6b and c, right). The nucleobase orien-
tation in 32 and 33 was akin to 21, that is, some population
of the syn conformation was present in acidic solution (Fig-
ures S15 and S16 in the Supporting Information). The
change in nucleobase conformation from anti to syn in
acidic solution can be rationalized on the basis of possible
electrostatic interactions between the positively charged pyr-
imidine ring and the negatively charged phosphate and/or
carboxylic acid groups.
In acidic solution, for deazaadenine-containing analogues
the syn conformers are detected, whereas this was not so for
the adenine analogue. This could be explained on the basis
of the higher nucleobase pKa values of the deazaadenines
compared with adenine, for which the existence of more
protonated forms is expected in acidic solutions. As dis-
cussed earlier, both the kinetic data and the molecular mod-
eling suggest the involvement of a protonated N3 atom in
3
tion, JH1’-H2’/H2’’ =7 Hz). In all of the Asp NPs, the nucleotide
part adopts the orientation (at both pD values) observed in
the nucleotide residue of regular DNA duplexes. The JC4’-P
(9.3 Hz) coupling constant was determined from the H-de-
3
1
coupled carbon spectrum, indicating that the b torsion angle
(C4’-C5’-O5’-P) is within the trans region.[38] Moreover, a
4
small JH4’-P coupling constant (ꢁ1–2 Hz) was detected by
comparing the 31P-coupled and -decoupled H4’ proton peak
(Figure S12b in the Supporting Information). This was fur-
ther confirmed by the appearance of a small cross peak in
the 1H–31P HETCOR spectra (Figure S12c in the Supporting
Information). The observation of this long-range coupling
(4JH4’-P) signified a planar W-shaped conformation of the mo-
lecular fragment H4’-C4’-C5’-O5’-P with a b torsion angle in
the anti region and g torsion angle (C3’-C4’-C5’-O5’) in the
gauche+ region.[38] The aspartyl moiety in all of these ana-
logues adopts a similar conformation at both pD values as
no significant change in the 3J homo- and heteronuclear cou-
pling constants was observed (Table S2 in the Supporting In-
formation). The aspartyl moiety was found to be close to
the sugar residue, irrespective of the solution pD, as shown
by the NOE correlation between the H3’ and Ha/Hb of Asp
that was observed in the ROESY spectra (Figure S13 in the
Supporting Information). This is somewhat in agreement
with the proposed molecular model, in which hydrogen
bonding between the b-COOH and the 3’-OH was shown.
To determine the existence of a syn conformation of the
nucleobase, as suggested by the molecular modeling study,
À
catalyzing the P O bond hydrolysis. The protonated N3
atom in adenine and 1- and 7-deazaadenine might be in-
volved in hydrogen bonding through a water molecule (or
directly) with the 5’-O of aaNPs, for which it can act to sta-
À
bilize the TS of the P O bond hydrolysis or by effectively
delivering a proton to the nucleoside leaving group through
the rare syn conformation. Based on the protonation site of
the nucleobase, the N3 protonated form of Asp-1-deaza-
dAMP is expected to be more abundant than for Asp-7-
deaza-dAMP and Asp-dAMP, for which the N3 protonated
form is rare because N1 is the predominant protonation site.
For Asp-3-deaza-dAMP, on the other hand, there is no pos-
sibility for N3 protonation. This may in general explain the
1H-coupled 13C, H–1H ROESY and H–31P HOESY experi-
ments were performed. The glycosyl torsion angle c was de-
1
1
3
3
fined by the JH1’-C4 (and JH1’-C8) coupling constant and based
on the H–1H and H–31P NOE interactions to discriminate
1
1
Chem. Eur. J. 2012, 18, 857 – 868
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