Contrasting behavior of conformationally locked carbocyclic nucleosides of adenosine and cytidine as substrates for deaminases

Article abstract of DOI:10.1080/15257770903091904

In addition to the already known differences between adenosine deaminase (ADA) and cytidine deaminase (CDA) in terms of their tertiary structure, the sphere of Zn+2 coordination, and their reverse stereochemical preference, we present evidence that the enzymes also differ significantly in terms of the North/South conformational preferences for their substrates and the extent to which the lack of the O(4') oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates. The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a rigid bicyclo[3.1.0]hexane scaffold.

Full text of DOI:10.1080/15257770903091904

Nucleosides, Nucleotides and Nucleic Acids, 28:614–632, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903091904
CONTRASTING BEHAVIOR OF CONFORMATIONALLY LOCKED
CARBOCYCLIC NUCLEOSIDES OF ADENOSINE AND CYTIDINE
AS SUBSTRATES FOR DEAMINASES
1
2
1
Victor E. Marquez, Gottfried K. Schroeder, Olaf R. Ludek,
1
1
2
Maqbool A. Siddiqui, Abdallah Ezzitouni, and Richard Wolfenden
1Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute
at Frederick, National Institutes of Health, Frederick, Maryland, USA
2
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill,
North Carolina, USA
2
In addition to the already known differences between adenosine deaminase (ADA) and cytidine
+2
deaminase (CDA) in terms of their tertiary structure, the sphere of Zn coordination, and their
reverse stereochemical preference, we present evidence that the enzymes also differ significantly in
terms of the North/South conformational preferences for their substrates and the extent to which the
ꢁ
lack of the O(4 ) oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates.
The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a
rigid bicyclo[3.1.0]hexane scaffold.
Keywords Adenosine deaminase; cytidine deaminase; carbocyclic nucleosides; bicy-
clo[3.1.0]hexane nucleosides
INTRODUCTION
Adenosine deaminase (ADA) and cytidine deaminase (CDA) catalyze
the deamination of adenosine and cytidine, respectively, via the formation
[
1,2]
of unstable hydrated intermediates.
ADA catalyzes the hydration of
the purine ring during the conversion of adenosine to inosine and CDA
catalyzes the hydration of the pyrimidine ring during the conversion of
Received 16 January 2009; accepted 1 June 2009.
In honor of and in celebration of Morris J. Robins’ 70th birthday.
This research was supported by the Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research and by grant GM-18325 (D.W. and G.K.S.). The authors thank Dr.
Megan Peach of the LMC for helping with the analysis and display of the crystal structures.
Address correspondence to Victor E. Marquez, Laboratory of Medicinal Chemistry, Center for
Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD,
21703, USA. E-mail: marquezv@dc37a.nci.nih.gov
6
14

Conformationally Locked Carbocyclic Nucleosides
615
FIGURE 1 Transition-state, hydrated intermediates formed during the enzymatic hydrolytic deamina-
tion of adenosine (A) and cytidine (B).
cytidine to uridine (Figure 1). Spontaneous deamination of the C4 posi-
tion of cytidine and of the C6 position of adenosine proceeds with rate
−
10 −1
constants around 10
s , whereas the enzymes are able to accelerate
[
3]
the rates of deamination by approximately 12–14 orders of magnitude.
These rate enhancements reflect extraordinary transition-state affinities
that are among the highest observed for any enzyme. In addition to
these important biochemical properties, ADA and CDA are of particular
interest as therapeutic targets for the treatment of several diseases, including
[
4–8]
cancer.
Formation of hydrated intermediates by these enzymes has been
confirmed by the crystal structures of inhibitors [nebularine (1a) and
zebularine analogues (2a,b)] with ADA and CDA, respectively. These crystal
structures revealed the formation of the 1,6-double bond hydrate (6-
hydroxyl-1,6-dihydropurine ribonucleoside) from nebularine and the 3,4-
double bond hydrate (4-hydroxy-3,4-dihydropyrimidine-2(1H)-one ribonu-
[
9–13]
cleoside) from zebularine) at the active sites.
In both enzymes, the
transition-state inhibitors replace the leaving NH group with a proton,
3
preventing conversion of the tetrahedral transition-state into product. Re-
markably, the presence of these hydrated species at the binding sites of the
enzymes suggest that the hydrated species may be associated with extraordi-
narily high enzyme inhibition affinities by resembling the transition states
of the deamination reactions.[
14]
Indeed, the inhibition constants for the
−
13
[1]
hydrated species of nebularine (1a, K = 10
M) and zebularine (2a, Ki
i
−
12
[2]
=
10
M) are quite remarkable. The true K ’s, however, are half of these
i
values since the addition of water to generate the tetrahedral intermediates
is stereospecific giving exclusively a single diastereoisomer (Figure 1). In
summary, these intermediates appear to capture much of the negative free

6
16
V. E. Marquez et al.
energy of binding expected of an ideal transition-state analogue for the
deamination reactions.
When the x-ray structures of ADA and CDA bound to the hydrates of
nebularine (1a) and zebularine (2a) are superimposed, it becomes clear
that these enzymes have opposite stereochemical preferences at the C6 (S)
[
15]
carbon for ADA and the C4 (R) carbon for CDA (Figures 1 and 2).
This
reverse stereochemical preference arises from the fact that the consensus
TVHA(G)E sequences of the two proteins curl around opposite sides of the
essential zinc. Therefore, because of this near mirror image relationship
of the two superimposed binding pockets formed around the zinc and
the conserved acid residues, these enzymes receive their substrates from
opposite sides.
Chemical changes in the aglycon of these inhibitors can affect the
formation of hydrated intermediates. For example, in the case of ADA,
the 100-fold increase in inhibitory potency exhibited by the 8-aza ana-
logue of nebularine (8-azapurine riboside, 1b) over nebularine itself is
probably related to the greater tendency of the 8-aza analogue to form a
[
16]
hydrate.
Similarly, in the case of CDA, fluorine substitution of zebularine
(
5-fluorozebularine, 2b), which is expected to promote hydration, helps
[
17]
explain the 10-fold increase in potency of 2b over zebularine.
The extent
of hydration of these modified bases and those of the natural substrates
is known to depend on protonation, which facilitates hydration relative to
the neutral species. Counter to the formation of the hydrated species is

Conformationally Locked Carbocyclic Nucleosides
617
FIGURE 2 (A) Bovine ADA in complex with hydrated nebularine.[11] Ligand is shown in ball-and-stick
representation, with green carbons. The crystal coordinates of residue His 14 are shown in dark gray. This
conformation is suggested to be an average of two rotamers: one (in green) where the Nδ atom makes a
bifurcated hydrogen bond to the sugar moiety in the ligand, and one (in light blue-gray) where the zinc
[
13]
atom lies in the plane of the imidazole ring. (B) E. coli CDA in complex with hydrated 5F-zebularine.
the loss in resonance energy resulting from hydration, which explains why
the existence of hydrated species free in solution are below the limits of
detection by direct methods.
In addition to the inverse mirror image relationship between ADA and
CDA, there are significant differences in the architecture of the enzyme
active sites that have important mechanistic implications: ADA is a monomer
+
2
and CDA is either a homodimer or a homotetramer. More critically, Zn
coordination in ADA is accomplished by five ligands (3 His, 1 Asp, and
a hydroxyl group from the transition-state inhibitor or a water molecule
[
9–11]
when bound to substrates) in a trigonal bipyramidal geometry.
In the
+
2
homodimeric E. coli CDA, Zn coordination is achieved with four ligands (2
Cys, 1 His and a hydroxyl group from the transition-state inhibitor or a water
[
13]
molecule when bound to substrates) in a nearly tetrahedral geometry.
+
2
On the other hand, the three Zn ligands in homotetrameric CDAs from B.
[
18]
[19]
[20]
subtilis,
mouse,
and human
are all cysteine residues. At the active
site environment of these enzymes, which is almost completely sequestered
+
2
from bulk solvent, the function of Zn is to activate a water molecule
to form a hydroxide ion for nucleophilic attack. Hence, the diverse co-
+
2
ordination spheres around Zn may affect the water/hydroxide equilib-
rium differently in each enzyme. In homotetrameric CDAs, the formal

6
18
V. E. Marquez et al.
negative charge of cysteinate increases the pK value of the bound water
a
−
and formation of the Zn-OH ion becomes less likely than if it were
coordinated to a tris-imidazolium zinc as in ADA. However, the excess
+
2
negative charge in the environment of the Zn in these homotetrameric
enzymes is compensated by an Arg residue (Arg56 in B. subtilis and Arg68 in
mouse and human CDAs), which lowers the pK for the zinc-bound water to
a
facilitate nucleophilic attack. In CDA, a key player for this concerted attack
is a conserved Glu residue (Glu104 in E. coli, Glu55 in B. subtilis, and Glu67
in mouse and human CDAs) which can act as (1) a general base to abstract a
proton from either zinc complex [Zn(Cys) (His)H O or Zn(Cys) H O], (2)
2
2
3
2
a general acid to protonate N3 of the pyrimidine ring, and (3) a shuttle for
[
181921]
proton transfer from the hydroxyl group to the departing ammonia.
In bovine ADA,[
11]
Glu 214 performs an equivalent role but there are
[
9,10,11,22]
other acidic residues near the active site of ADA (Figure 2A).
Asp293, for example (not shown), which is in the vicinity of hydrophobic
residues, serves as a hydrogen bond donor to the lone pair of the very
basic nitrogen N7. These differences could be important when considering
[
23]
[23]
the pK of adenosine (pK = 3.5)
and cytidine (pK = 4.2)
and
a
a
a
their ease of protonation inside the folded structures of ADA and CDA,
which can perturb the active site to allow protonation near physiological
pH. Intuitively, it should be easier to protonate cytidine than adenosine
and that is probably why there are more acidic residues in ADA (Asp16,
Glu214, Asp292 and Asp293 in bovine ADA) compared to a solitary Glu in
CDA (Glu104 in E. coli, Glu55 in B. subtilis, and Glu67 in mouse and human
CDAs). Perhaps too that is the reason why ADA binds to adenosine in a
-endo, North conformation (vide infra).
The sugar moiety, has a small effect on the rate of spontaneous deamina-
tion but greatly enhances the reactivity of both ADA and CDA by increasing
ꢁ
[22]
3
[
24,25]
the effective concentration of the altered substrate at the active site.
This entropic advantage is the direct result of the parts (base and ribose)
being properly connected to fit the active site. As far as a direct contribution
to catalysis, however, because the sugar ring is distant from the site of the
chemical transformation, its role has been considered to be insignificant or
at best modest.[
26,27]
In contrast, studies in our laboratory with carbocyclic
nucleosides have revealed that the sugar moiety in ADA seems to play a
significant role in catalysis by providing effective electronic communication
ꢁ
between the O(4 ) and the base through the anomeric effect and also by
[
28]
providing electron-withdrawing activation.
In addition, the crystal structure of the transition-state inhibitor bound
to ADA shows that His 14, one of the Zn ligands, may interact strongly
with O(4 ) and with the 5 -OH of the sugar (Figure 2A). Therefore, the
loss of this interaction resulting from the replacement of the furanose ring
of adenosine or 2 -deoxyadenosine with a cyclopentane ring —as in the
+
2
ꢁ
ꢁ
ꢁ
fermentation product aristeromycin (3)— also helps explains the significant

Conformationally Locked Carbocyclic Nucleosides
619
[
28]
drop in the deamination rate of 3 to 0.58% the rate of adenosine.
ꢁ
Obviously the absence of the O(4 ) oxygen abolishes the stereoelectronic
interplay between the purine ring and the ribofuranosyl moiety, impacting
[
29]
negatively on the propensity of the base to form a covalent hydrate.
It
has been clearly demonstrated by Chattopadhyaya and coworkers that the
electronic properties of the aglycon are effectively transmitted to the pento-
[
29,30]
furanose moiety through the anomeric effect.
Because the anomeric
effect is more effectively transmitted when the conformation of the sugar is
ꢁ
North, protonation of adenosine and 2 -deoxyadenosine is more effective in
the North conformation and therefore it is not surprising that in the crystal
structures of ADA in complex with 6-hydroxyl-1,6-dihydropurine ribonucle-
ꢁ
[22]
oside, the ribose conformation is in a 3 -endo (North) ring pucker.
In
ꢁ
the North conformation, the equatorial 3 -hydroxyl group of the ribose is
ꢁ
engaged in hydrogen bonding with Asp16, whereas the 2 -hydroxyl does
[
9–11,22]
not appear to play an essential role.
This finding agrees with the
ꢁ
fact that 2 -deoxyadenosine is as good a substrate for ADA as adenosine.
Furthermore, in the North conformation Glu214 is perfectly aligned to
donate a proton to N1 of the purine ring, thereby making the C6 carbon
more susceptible to hydration (Figure 2A).
ꢁ
As expected, the absence of the O(4 ) oxygen also caused a large decline
in the deamination rates of two conformationally locked North-dAdo (4) and
South-dAdo (5) carbocyclic nucleosides to 1% and 0.01%, respectively, of the
[
28]
values observed for adenosine.
Significantly, despite this considerable
drop in activity, the enzyme still showed a 100-fold preference for North-
dAdo (4) over South-dAdo (5), and the former compound was in turn
preferred by nearly 2-fold over the flexible aristeromycin (3). Based on
these findings we postulated that the preference of ADA for North-dAdo
ꢁ
resulted principally from the correct equatorial disposition of the 3 -OH
engaged in effective hydrogen bonding with Asp16, as observed in the
crystal structure.[ In summary, the combined factors responsible for the
low deamination rate of carbocyclic adenosine nucleosides are the reduced
11]
ꢁ
level of hydration of the base and the missing interaction of the O(4 )
ꢁ
oxygen with His 14. The removal of the O(4 ) oxygen appears to be so
critical that even a North-locked version of the 8-azaadenosine failed to show
a comparable enhancement in the rate of deamination as it was observed
for the corresponding nucleoside.[
31]
Intriguingly, CDA has a totally different preference for the
sugar pucker of substrates and inhibitors, and, as opposed to ADA,
ꢁ
ꢁ
the ribose and 2 -deoxyribose glycons adopt the antipodal 2 -endo
South conformation.[
13,18–20]
In the crystal structure of hydrated
5
3
-fluorozebularine (2b) at the active site of E. coli CDA, it is the axial
ꢁ
ꢁ
-OH that engages in hydrogen bonding with Glu91, while the 2 -OH does
[
13]
not interact with the protein (Figure 2B).
the 2 -OH is in agreement with the known experimental fact that cytidine
The lack of involvement of
ꢁ

6
20
V. E. Marquez et al.
ꢁ
and 2 -deoxycytidine are equally good substrates for CDA. Also, in the South
conformation, Glu104 (E.coli CDA) is in perfect alignment to provide the
[
13]
critical protonation at N3 of the pyrimidine ring.
More importantly,
because of the mirror image relationship of the binding pockets of ADA
ꢁ
and CDA, the ribose (or 2 -deoxyribose) ring of the substrates are in
opposite orientations relative to the zinc’s coordination sphere; in the case
ꢁ
of ADA, His 14 can interact with O(4 ) (Figure 2A), whereas in the case of
CDA from E. Coli a similar interaction with His 102 is not possible (Figure
2
B). Furthermore, other CDAs have all cysteine residues around the zinc.
In the present investigation we confirm ADA’s preference for North
conformer substrates by utilizing the conformationally locked carbocyclic
nucleosides North-dAdo (4) and South-dAdo (5). We demonstrate the an-
tipodal preference of CDA for South conformer substrates employing a
similar approach with an equivalent set of carbocyclic nucleosides having
a flexible cyclopentane ring (carbodine, 6), or conformationally locked
North and South versions of cytidine [North-dCyd (7) and South-dCyd (8)]
[
32,33]
built on a bicyclo[3.1.0]hexane platform.
We also highlight how the
important architectural differences between ADA and CDA can help explain
ꢁ
the changes in catalysis observed in going from ribose (or 2 -deoxyribose)
nucleosides to their carbocyclic isosteres in these two mechanistically similar
enzymes.
EXPERIMENTAL RESULTS
Kinetic Analysis of Locked Carbocyclic Adenosine Analogues,
North-dAdo (4) and South-dAdo (5), as Substrates of ADA
ADA activity was measured spectrophotometrically as described for
the natural substrate adenosine following the decrease in absorbance at
[
34]
264 nm.
An initial comparison between North-dAdo and South-dAdo
analogues (Figure 3) was performed using ADA from bovine spleen (bs).
◦
The experiments were run in 0.1 M phosphate buffer (pH 7.4) at 25 C using
60 µM substrate (essentially identical to conditions for CDA studies, vide
infra).

Conformationally Locked Carbocyclic Nucleosides
621
FIGURE 3 Bovine spleen ADA (∼0.5 units) deamination of South-dAdo (ꢀ) and North-dAdo (◦) at 60
◦
µM determined by measuring the decrease in absorbance at 264 nm (25 C).
Relative rates (Table 1) were calculated using initial rates following
the first ∼10% of reaction (Figures 1S and 2S, Supporting Information).
◦
◦
Despite the difference in temperature (37 C vs. 25 C) and substrate con-
centration (50 µM vs. 60 µM) used in these experiments, compared to
our previous published results,[
28]
the conclusion is the same: the North
analogue is clearly a better substrate than the South analogue by more than
two orders of magnitude (Figure 2, Table 1). Table 1 also shows that the two
ADA enzymes from calf-intestine (ci) and bovine serum (bs) behaved very
similarly toward these substrates.
Due to the relatively long incubation time of South-dAdo with ADA (∼22
hours), an enzyme control (same conditions) was monitored for activity loss
TABLE 1 Relative rates for the North-dAdo and South-dAdo analogues compared to the
normal ADA substrates with ADA from calf-intestine (ci) and bovine serum (bs)
ADA (ci)[
28]
(50
ADA (ci) (60 µM,
25 C)
Rel. Rates
ADA (bs) (60 µM,
25 C)
Rel. Rates
◦
◦
◦
µM, 37 C)
Rel. Rates
Ado
100
100
100
dAdo
121
124
119
North-dAdo
South-dAdo
0.990
0.010
1.210
0.007
1.380
0.008

6
22
V. E. Marquez et al.
FIGURE 4 Full UV spectrum for the enzymatic deamination of North- and South-dCyd over time. A clean
isobestic point at 250 nm is maintained throughout the course of the reaction. The inset corresponds to
the drop in absorbance at 284 nm (long arrow) as a function of time. The short arrows indicate either
the increase (↑) or decrease (↓) in absorbance at that point over time. A) North-dCyd (ꢁ). B) South-dCyd
(
ꢂ).
during the course of the reaction. This sample showed no significant change
◦
in activity when compared with a second enzyme control kept at 4 C.
ꢁ
From the data in Table 1 it is also clear that the effect of the 4 (O) is
critical for the ADA reaction. However, despite the >100-fold difference

Conformationally Locked Carbocyclic Nucleosides
623
FIGURE 5 Initial time course data for the CDA (900 nM) catalyzed deamination of North-dCyd (ꢁ)
2
84
and South-dCyd (ꢂ) followed at 284 nm (ꢀε
= −5000). The inset shows initial time course data for
the catalyzed deamination of the natural substrate cytidine (ꢃ) by CDA (0.9 nM) monitored at 282 nm
282
(
ꢀε
= –3600).
between the natural substrates and the two locked analogues, ADA shows
a definitive preference for the North conformation, in agreement with
the conformation of the sugar ring observed in the crystal structures of
inhibitors bound to ADA.[
9–11,22]
FIGURE 6 Michaelis-Menten plot for enzyme catalyzed deamination of cytidine (ꢃ) and carbodine
(
ꢄ). The solid line represents a non-linear regression fit of the cytidine data to the Michaelis-Menten
equation.

6
24
V. E. Marquez et al.
Kinetic Analysis of Locked Carbocyclic Cytidine Analogues,
North-dCyd (7) and South-dCyd (8), as Substrates of CDA
Cytidine deaminase activity was measured spectrophotometrically as
2
82
[35]
described for the natural substrate cytidine (ꢀε
= −3600).
The
ꢁ
280
deamination of 2 -deoxycytidine (dCyd) was monitored at 280 nm (ꢀε
5000; difference in extinction coefficients between dCyd and 2 -
deoxyuridine at 280 nm ). The extinction coefficients for the locked
North- and South-dCyd derivatives were based on that of 2 -deoxycytidine
=
ꢁ
−
[
36]
ꢁ
max
272
[36]
(
λ
= 272, ε = 9000 ). However, a comparison of the UV spectrum
of dCyd with that of South-dCyd (8) revealed a complete 4 nm shift to
longer wavelengths in the region of 270 to 290 nm (Figure 3S, Support-
ing Information). Therefore, the enzymatic deamination of both locked
derivatives was monitored by the decrease in absorbance at 284 nm rather
2
84
max
than 280 nm (ꢀε = −5000). The λ
was also correspondingly shifted
max
276
by 4 nm (λ
= 276, ε
= 9000). Because the locked compounds were
such poor substrates, it was necessary to use a higher concentration of
enzyme and follow the reaction for longer periods of time to determine
their deamination rates relative to cytidine.
The deamination of both North-dCyd (62 µM) and South-dCyd (61 µM)
was followed over the course of two days at a CDA concentration of ∼900
nM in phosphate buffer (100 mM) at pH 6.8. Controls containing the
analogues in the absence of enzyme showed no change over the same time
period. UV spectra of enzyme/buffer control samples were subtracted from
all raw spectra of the actual CDA/analogue samples. All spectra represent
an average of 5 sequential runs (Figure 4). The deamination of both
compounds proceeded with a single isobestic point at 250 nm, indicative of
direct enzymatic conversion of substrate to product. It is also clear that while
the deamination of South-dCyd was complete well within the experimental
time frame (48 hours), the deamination of North-dCyd had not yet reached
completion (see insets).
Initial rates for CDA (900 nM) catalyzed deamination of South-dCyd (ꢀ)
and North-Cyd (ꢁ), each at 70 µM, were also determined by a linear fit to
initial rate data (Figure 5, Table 2). The inset shows the initial rate data for
the CDA (0.9 nM) catalyzed deamination of the natural substrate cytidine
(
ꢂ), also at 70 µM. This data shows that the deamination of South-dCyd is
roughly an order of magnitude faster than that of North-dCyd.
Kinetic Analysis of Plain Carbocyclic Cytidine (Carbodine, 8) as a
Substrate of CDA
Concentrations of the plain carbocyclic cytidine analogue (carbodine)
max
275
[37]
were measured at λ , 275 nm (ε = 9300 ), and enzymatic deamina-
tion was monitored by the increase in absorbance at 267 nm for the uridine

Conformationally Locked Carbocyclic Nucleosides
625
TABLE 2 Relative deamination rates for carbocyclic
cytidine derivatives
Substrate
Relative Rates
dCyd
Cytidine
270
100
Carbodine (6)
South-dCyd (8)
North-dCyd (7)
25
0.001
0.0001
analogue (ꢀε267 = 2800). The value for ꢀε
267
was calculated using the
experimentally determined extinction coefficient for carbodine at 267 nm
(
2
ε = 7900) and the reported extinction coefficient for carbocylic uridine at
[
38]
67 nm (ε = 10,700 ).
Figure 6 shows a Michaelis-Menten plot for both cytidine and carbodine.
Interestingly, initial rates for carbodine deamination were only ∼4-fold
lower than those of cytidine over the observed substrate concentration
range (Table 2). At higher concentrations of carbodine, there appeared to
be a slight decrease in enzymatic activity, but the source of this deviation was
not investigated further.
When the relative rates for all five compounds determined at 70 µM
were adjusted for differences in enzyme concentration (Table 2) it can be
appreciated that the flexibility of the cyclopentane in carbodine allows it
to be a better substrate for CDA. In fact, the best substrate of the locked
analogues (South-dCyd) is still dismally poor relative to carbodine despite
having the required South conformation. These preferences are definitely
different from those of ADA where the flexible, carbocyclic analogue
(aristeromycin, 3) was intermediate between the better North-dAdo and
poorer South-dAdo substrates. It may also be worth noting that dCyd is a
[
39]
slightly better substrate than cytidine (kcat ∼3-fold faster ), consistent with
our results (Table 2).
DISCUSSION
In addition to the structural differences between ADA and CDA that
were discussed earlier, and their opposite stereochemical preferences in
forming the hydrated intermediates, our studies confirm that the con-
formational preferences of ADA and CDA for substrates built as locked
carbocyclic nucleosides are also opposite between North and South. The
preferences that we have determined agree well with the conformations
of conventional nucleosides, substrate or inhibitors, observed in the crystal
structures of the complexes. However, in terms of reactivity, the differences
that separate these two seemingly similar enzymes are staggering. For ADA,
the North-South-dAdo analogues mimic the natural substrates better than

6
26
V. E. Marquez et al.
the North-South-dCyd analogues are able to mimic the natural substrates for
CDA. In other words, the North-South-dAdo analogues are better substrates
for ADA than the North-South-dCyd analogues are for CDA. We know this by
comparing how well these analogues work relative to the normal substrates:
North-dAdo is only ∼100 times worse than adenosine, but South-dCyd is
nearly ∼100,000 times worse than cytidine. In each case, we are making a
comparison to the normal substrate, so we are addressing structural differ-
ences between the normal substrate and the carbocyclic substrates for their
respective enzymes. For both ADA and CDA it is clear that the removal of
ꢁ
the O(4 ) oxygen reduces the capacity of the substrate to undergo efficient
deamination. However, why are the two enzymes so different in terms of
ꢁ
the impact of the missing O(4 )? In the case of ADA, despite the ∼100-fold
drop in the rate of deamination caused by the missing oxygen, the enzyme
clearly prefers North-dAdo (4) over South-dAdo (5). In view of the absence
ꢁ
of stereoelectronic effects and the key interaction between O(4 ) and His
1
4, the selection of North-dAdo as the better substrate by ADA appears to
be driven exclusively by the North conformation of the locked sugar and its
ability to reproduce the important network of hydrogen bonds seen with
ꢁ
the natural substrates bound in the 3 -endo, North conformation. All things
ꢁ
being equal in terms of the missing O(4 )-effect, the rate of deamination
of the conformationally flexible aristeromycin (3), lies between the rates
of deamination of the rigid North-dAdo and rigid South-dAdo and supports
ADA’s preference for the North conformer.
The situation with CDA is completely different. Not only CDA has
the opposite preference for the South conformer (which it deaminates
∼
100,000-fold less efficiently than cytidine), as inferred from the 10-fold
faster rate of deamination of South-dCyd (8) over North-dCyd (7) (Table 2),
but also the flexible carbocyclic carbodine (6) is a very good substrate, just
4
-fold less efficient than cytidine!
Because formation of the hydrated intermediates is key for the deamina-
tion reactions to go forward efficiently, we need to address the impact that
ꢁ
the sugar puckering and the O(4 ) have on the protonation of the substrates
−
which is an important step prior to the nucleophilic attack by Zn-OH at the
active site in both enzymes. In order to explain these differences, we propose
the following arguments:
1
. The pK of adenosine (pK = 3.5) is 0.7 log units lower than that of
a
a
cytidine (pK = 4.2) and hence it is much harder to protonate. This is
a
probably why there are more acidic residues in ADA (Glu214, Asp292,
and Asp293) in direct contact with the base.
ꢁ
2
. The O(4 ) anomeric effect is more effectively transmitted when the
conformation of the sugar is North; therefore, protonation of the base

Conformationally Locked Carbocyclic Nucleosides
627
ꢁ
in adenosine and 2 -deoxyadenosine is facilitated when both nucleosides
adopt the North conformation at the active site of ADA.
ꢁ
3
4
. The influence of the O(4 ) anomeric effect in driving the conformation
ꢁ
of 2 -deoxyadenosine to the North at the active site of ADA has to be
critical because this nucleoside is more stable in the South conformation
due to the gauche effect between the 3 -OH and O(4 ).
ꢁ
ꢁ
[29]
ꢁ
. In the absence of O(4 ), the combined loss of anomeric effect and the
lack of interaction with His 14 significantly reduces the deamination rate
of carbocyclic adenosine analogues by ADA, and the preference for the
North substrate is driven solely by the shape of the binding pocked of
ADA which accommodates the North conformer more effectively.
. Aristeromycin (3) has a ring pucker that favors a conformation away from
the North[ orientation and hence there is an additional penalty to shift
the flexible cyclopentane ring to the North conformation required by
ADA.
5
40]
6
. The pK of cytidine is higher, and a single solitary Glu residue in CDA is
a
able to perform the protonation of the base.
ꢁ
7
. Because of the higher pK of cytidine, the O(4 ) anomeric effect may
a
not be critical in facilitating the protonation of cytosine at the active site
ꢁ
ꢁ
ꢁ
of CDA. The 3 -OH/O(4 ) gauche effect, particularly in 2 -deoxycytidine,
[
28]
drives the conformation to the South
to which CDA can bind effec-
tively.
ꢁ
8
9
. The diminished role of the O(4 ) anomeric effect facilitating protonation
of cytidine substrates by CDA agrees with the fact the flexible carbocyclic
nucleoside carbodine (6) is a good substrate. Protonation of carbodine
is even easier due to its higher pK , which is ∼0.45 log units higher than
a
native cytidine nucleosides.[ Therefore, the only penalty paid during
the binding of carbodine at the active site of CDA is that of changing its
flexible conformation[ to the South.
41]
42]
. According to the crystal structure of CDA bound to either substrates
ꢁ
or inhibitors, there is no interaction between the O(4 ) oxygen of the
sugar with the ligands around the zinc’s coordinating sphere. Therefore,
ꢁ
changing from ribose (or 2 -deoxyribose) to cyclopentane is not as
important and the small decrease in the deamination rate of carbodine
reflects only the absence of anomeric effect and its role in facilitating
hydration.
10. South-dCyd (8) is a poorer substrate relative to carbodine (6) because
of the well known tendency in this class of South bicyclo[3.1.0]hexane
nucleosides to force the base into the more stable, unnatural syn
conformation.[
43]
This conformation is further stabilized by an in-
ꢁ
tramolecular hydrogen bond between the 5 -OH and the pyrimidine
carbonyl in a binding pocket sequestered from bulk water. The fusion of
the cyclopropane ring adjacent to the C N bond significantly increases
the barrier of rotation around the glycosyl bond and the ensuing penalty

6
28
V. E. Marquez et al.
to rotate the glycosyl bond into the anti range to fit the enzyme is
[
43]
severe.
1
1. Despite the severity of the penalty that the South-dCyd substrate must
pay to rotate the cytosine base into the required anti range, the enzyme
still recognizes the South substrate more effectively than the North coun-
[
43]
terpart even when the latter favors the anti conformation.
The North
conformer simply has the wrong sugar pucker and does not fit effectively
at the active site of CDA.
1
2. The behavior of carbodine as a good substrate for CDA agrees with
previous studies form our laboratory showing that the carbocyclic ana-
logue of zebularine experienced only 16-fold drop in potency as a CDA
inhibitor.[
45]
In summary, we believe that the staggering differences in the rates of
deamination of carbocyclic analogues of adenosine and cytidine by ADA
and CDA are the result of complex, antipodal stereoelectronic interactions
that further confirm the lack of evolutionary homology between them.
EXPERIMENTAL SECTION
Enzyme Assays
ꢁ
Cytidine, 2 -deoxycytidine, potassium phosphate (monobasic and diba-
sic) and adenosine deaminase (bovine spleen and calf intestinal) were
obtained from Sigma. Cytidine deaminase was purified as previously
[
34]
◦
described.
All assays were performed in 1 cm quartz cuvettes at 25 C in
0.1 M phosphate buffer (pH 6.8) on an HP 8452a diode array spectropho-
tometer. Relative kinetic rates were determined using initial rates (following
the first ∼10% of reaction), corrected for any differences in enzyme concen-
tration. Substrate concentrations were either 60 µM (adenosine analogues)
or 70 µM (cytidine analogues). Deamination of substrate analogues (60
µM) were also followed to completion, where possible. Control experiments
were also run to test the stability of the analogues and the enzyme solutions
individually, under the same conditions. All of the other experimental
information is included in the main text under results, which was necessary
to help interpret the figures.
REFERENCES
1
. Jones, W.; Kurz, L.C.; Wolfenden, R. Transition-state stabilization by adenosine deaminase: 1,6-
addition of water to the purine ribonucleoside, the enzyme’s affinity for 6-hydroxy-1,6-dihydropurine
ribonucleoside, and the effective concentration of substrate water at the active site. Biochemistry,
1989, 28, 1242–1247.
2
. Frick, L.; Yang, C.; Marquez, V.E.; Wolfenden, R. Binding of pyrimidin-2-one ribonucleoside by
cytidine deaminase as the transition-state analogue of 3,4-dihydrouridine and the contribution of
the 4-hydroxyl group to its binding affinity. Biochemistry 1989, 28, 9423–9430.

Conformationally Locked Carbocyclic Nucleosides
629
3. Schroeder, G.K.; Wolfenden, R. Rates of spontaneous disintegration of DNA and the rate
enhancements produced by DNA glycosylases and deaminases. Biochemistry, 2007, 46, 13638–
13647.
4
5
6
7
. Agarwal, R.P.; Spector, T.; Parks, R.E. Tight-binding inhibitors—IV. Inhibition of adenosine deami-
nases by various inhibitors. Biochem. Pharmacol. 1979, 26, 259–267.
. Glazer, R.I. Adenosine deaminase inhibitors: Their role in chemotherapy and immunosuppression.
Cancer Chemother. Pharmacol. 1980, 4, 227–235.
. Smyth, J.F.; Prentice, H.G.; Proctor, S.; Hoffbrand, A.V. Deoxycoformycin in the Treatment of
Leukemias and Lymphomasa. Ann. N.Y. Acad. Sci. 1985, 451, 123–128.
. Laliberte, J.; Marquez, V.E.; Momparler, R.L. Potent inhibitors for the deamination of cytosine
ꢁ
arabinoside and 5-aza-2 -deoxycytidine by human cytidine deaminase. Cancer Chemother. Pharmacol.
1992, 30, 7–11.
8
9
. Lemaire, M.; Momparler, L.F.; Bernstein, M.; Marquez, V.E.; Momparler, R.L. Enhancement of
antineoplastica acion of 5-aza-2 -deoxycytidine by zebularine on L1210 leukemia. Anti-Cancer Drugs.
ꢁ
2005, 16, 301–308.
. Wilson, D.K.; Rudolph, F.B.; Quiocho, F.A. Atomic structure of adenosine deaminase complexed
with transition-state analog: understanding catalysis and immunodeficiency mutations. Science 1991,
252, 1278–1284.
1
1
1
1
1
0. Sharff, A.J.; Wilson, D.K.; Chang. Z.; Quiocho, F.A. Refined 2.5 A˚ structure of murine adenosine
deaminase at pH 6.0. J. Mol. Biol. 1992, 226, 917–921.
1. Kinoshita, T.; Nishio, N.; Nakanishi, I.; Sato, A.; Fujii, T. Structure of bovine adenosine deaminase
complexed with 6-hydroxy-1,6-dihydropurine. Acta Cryst. 2003, D59, 299–303.
2. Xiang, S.; Short, S.A.; Wolfenden, R.; Carter, C.W. Jr. Transition-state selectivity for a single hydroxyl
group during catalysis by cytidine deaminase. Biochemistry 1995, 34, 4516–4523.
3. Betts, L.; Xiang, S.; Short, S.A.; Wolfenden, R.; Carter, C.W. Jr. Cytidine deaminase. The 2.3 A˚ crystal
structure of an enzyme:transition-state complex. J. Mol. Biol. 1994, 235, 635–656.
4. According to high level calculations it appears that the zebularine 3,4-hydrate may be unstable and
that proton transfer from the Zn-OH group to Glu-104 in E. coli leads to an alkoxide-like inhibitor
that functions a the real tansition state. Guo, H.; Rao, N.; Xu, Q.; Guo, H. Origin of tight binding of
a near-perfect transition-state analogue by cytidine deaminase: Implications for enzyme catalysis. J.
Am. Chem. Soc. 2005, 127, 3191–3197.
15. Carter, C.W. Jr. The nucleoside deaminases for cytidine and adenosine: structure, transition state
stabilization, mechanism, and evolution. Biochimie 1995, 77, 92–98.
16. Seewach, D.S.; Krawczyk, S.H.; Acevedo, O.L.; Townsend, L.B. Rapid communication: Inhibi-
tion of adenosine deaminase by azapurine ribonucleosides. Biochem. Pharmacol. 1992, 44, 1697–
1700.
1
1
1
7. McCormack, J.J.; Marquez, V.E.; Liu, P.S.; Vistica, D.T.; Driscoll, J.S. Inhibition of cytidine deaminase
by 2-oxopyrimidine riboside and related compounds. Biochem. Pharmacol. 1980, 29, 830–832.
8. Johansson, E.; Mejlhede, N.; Neuhard, J.; Larsen, S. Crystal structure of the tetrameric cytidine
deaminase from Bacillus subtilis at 2.0 A˚ resolution. Biochemistry, 2002, 41, 2563–2570.
9. Teh, A.-H.; Kimura, M.; Yamamoto, M.; Tanaka, N.; Yamaguchi, I.; Kumasaka, T. The 1.48 A˚
resolution crystal structure of the homotetrameric cytidine deaminase from mouse. Biochemistry
2006, 45, 7825–7833.
20. Chung, S.; Fromme, J.C.; Verdine, G.L. Structure of human cytidine deaminasebound to a potent
inhibitor. J. Med. Chem. 2005, 48, 658–660.
2
1. Carlow, D.C.; Carter, C.W. Jr.; Mejlhede, N.; Neuhard, J.; Wolfenden, R. Cytidine deaminase from B.
subtilis and E. coli: Compensating effects of changing zinc coordination and quaternary structure.
Biochemistry 1999, 38, 12258–12265.
22. Wang, Z.; Quiocho, F.A. Complexes of adenosine deaminase with two potent inhibitors: X-ray
structures in four independent molecules at pH of maximum activity. Biochemistry, 1998, 37,
8314–8324.
2
2
3. See http://www.steve.gb.com/science/nucleic acids.html for listings of nucleoside pKa’s
4. Kati, W.M.; Acheson, S.A.; Wolfenden, R. A transition state in pieces: Major contributions of entropic
effects to ligand binding by adenosine deaminase. Biochemistry, 1992, 31, 7356–7366.
5. Carlow, D.; Wolfenden, R. Substrate connectivity in the transition state for cytidine deaminase.
Biochemistry 1998, 37, 11873–11878.
2

6
30
V. E. Marquez et al.
2
6. Erion, M.D.; Reddy, M.R. Calculation of relative hydration free energy differences for heteroaro-
matic compounds: use in the design of adenosine deaminase and cytidine deaminase inhibitors. J.
Am. Chem. Soc. 1998, 120, 3295–3304.
2
7. Carlow, D.C.; Short, S.A.; Wolfenden, R. Complementary truncations of a hydrogen bond to ribose
involved in transition-state stabilization by cytidine deaminase. Biochemistry, 1998, 37, 1199–1203.
8. Marquez, V.E.; Russ, P.; Alonso, R.; Siddiqui, M.A.; Hernandez, S.; et al. Synthesis of conformationally
restricted carbocyclic nucleosides. The role of the O4’-oxygen in the key hydration step of adenosine
deaminase. Helv. Chim. Acta, 1999, 82, 2119–2129.
2
29. Plavec, J.; Tong, W.; Chattopadhyaya, J. How do gauche and anomeric effects drive the pseu-
dorotational equilibrium of the pentofuranose moiety of nucleosides? J. Am. Chem. Soc. 1993, 115,
9734–9746.
3
0. Thibaudeau, C.; Plavec, J.; Chattopadhyaya; J. Quantitation of the pD dependent thermodynamics
of the NDS pseudorotational equilibrium of the pentofuranose moiety in nucleosides gives a direct
measurement of the strength of the tunable anomeric effect and the pKa of the nucleobase. J. Org.
Chem. 1996, 61, 266–286.
3
1. Hernandez, S.; Ford, H. Jr.; Marquez, V.E. Is the anomeric effect an important factor in the
rate of adenosine deaminase catalyzed hydrolysis of purine nucleosides? A direct comparison of
nucleoside analogues constructed on ribose and carbocyclic templates with equivalent heterocyclic
bases selected to promote protonation. Bioorg. Med. Chem. 2002, 10, 2723–2730.
3
2. Marquez, V.E.; Siddiqui, M.A.; Ezzitouni, A.; Russ, P.L.; Wang, J.; et al. Nucleosides with a twist. Can
fixed forms of sugar ring pucker influence biological activity in nucleosides and oligonucleotides? J.
Med. Chem. 1996, 39, 3739–3747.
3
3. Ezzitouni, A.; Barchi Jr., J.J.; Marquez, V.E. Conformationally locked carbocyclic nucleosides built on
a bicyclo[3.1.0]hexane template with a fixed southern conformation. Synthesis and antiviral activity.
J. Chem. Soc. Perkin Trans 1 1997, 1073–1078.
34. Cercignani, G.; Allegrini, S. On the validity of continuous spectrophotometric assays for adenosine
deaminase activity: A critical reappraisal. Anal. Biochem. 1991, 192, 312–315.
35. Cohen, R.M.; Wolfenden, R. Cytidine deaminase from Escherichia coli: Purification, properties and
inhibition by the potential transition state analog 3,4,5,6-tetrahydrouridine. J. Biol. Chem. 1971, 246,
7561–7565.
3
3
3
3
4
6. Sober, H.A. Handbook of Biochemistry: Selected Data for Molecular Biology, ed. H.A. Sober, Chemical
Rubber Company, Cleveland, OH, 1968, pp. G46–G50,
7. Shealy, Y.F.; O’Dell, C.A. Carbocyclic analogs of cytosine nucleosides. J. Heterocycl. Chem. 1980, 17,
3
53–358.
8. Shealy, Y.F.; O’Dell, C.A. Synthesis of carbocyclic analogs of uracil nucleosides. J. Heterocycl. Chem.
976, 13, 1015–1020.
1
9. Ashley, G.W.; Bartlett, P.A. Purification and properties of cytidine deaminase from Escherichia coli.
J. Biol. Chem. 1984, 259, 13615–13620.
0. Thibaudeau, C.; Kumar, A.; Bekiroglu, S.; Matsuda, A.; Marquez, V.E.; Chattopadhyaya, J. NMR
ꢁ
ꢁ
conformation of (-)-beta-D-aristeromycin and its 2 -deoxy and 3 -deoxy counterparts in aqueous
solution. J. Org. Chem. 1998, 63, 5447–5462.
4
1. Froehler, B.C.; Ricca, D.J. Triple helix formation by oligodeoxynucleotides containing the carbo-
ꢁ
cyclic analogs of thymidine and 5-methyl-2 -deoxycytidine. J. Am. Chem. Soc. 1992, 114, 8320–8322.
42. Although there is no crystal structure of (-)-carbodine, we could assume that its conformation
mirrors that of aristeromycin which in the solid state is almost a perfect East conformation (P =
◦
◦
◦
◦
◦
8
9 ) and in solution it is in a dynamic equilibrium between 35 < P < 65 and 128 < P < 131 (ref.
40).
4
3. Marquez, V.E.; Ben-Kasus, T.; Barchi, J.J. Jr.; Green, K.M.; Nicklaus, M.C.; Agbaria, R. Experimental
and structural evidence that herpes 1 kinase and cellular DNA polymerase(s) discriminate on the
basis of sugar pucker. J. Am. Chem. Soc. 2004, 126, 543–549.
44. Jeong, L.S.; Buenger, G.; McCormack, J.J.; Cooney, D.A.; Hao, Z.; Marquez, V.E. Carbocyclic
analogues of the potent cytidine deaminase inhibitor 1-(ß-D-ribofuranosyl)-1,2-dihydropyrimidine-
2
-one (Zebularine). J. Med. Chem. 1998, 41, 2572–2578.

Conformationally Locked Carbocyclic Nucleosides
SUPPLEMENTARY INFORMATION
631
FIGURE 1S Initial rate for the ADA (bovine spleen) catalyzed deamination of South-dAdo (ꢀ) at 60 µM
◦
and 25 C.
FIGURE 2S Initial rate for the ADA (bovine spleen) catalyzed deamination of North-dAdo (◦) at 60 µM
◦
and 25 C.

6
32
V. E. Marquez et al.
ꢁ
FIGURE 3S UV spectrum for 2 -deoxycytidine (dashed line) and South-dCyd (solid line). The UV
ꢁ
spectrum of 2 -deoxycytidine, shifted by 4 nm is included for reference (dotted line).