Molecular recognition of adenosine deaminase: 15N NMR studies

Article abstract of DOI:10.1080/15257770601112713

The elucidation of the molecular recognition of adenosine deaminase (ADA), the interpretation of the catalytic mechanism, and the design of novel inhibitors are based mostly on data obtained for the crystalline state of the enzyme. To obtain evidence for

Full text of DOI:10.1080/15257770601112713

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Molecular Recognition of Adenosine
Deaminase: 15N NMR Studies
Avital Laxer a , Hugo E. Gottlieb a & Bilha Fischer a
a Department of Chemistry , Gonda-Goldschmied Medical Research
Center, Bar-Ilan University , Ramat-Gan, Israel
Published online: 24 Jan 2007.
To cite this article: Avital Laxer , Hugo E. Gottlieb & Bilha Fischer (2007) Molecular Recognition of
Adenosine Deaminase: 15N NMR Studies, Nucleosides, Nucleotides and Nucleic Acids, 26:2, 161-180,
DOI: 10.1080/15257770601112713
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Nucleosides, Nucleotides, and Nucleic Acids, 26:161–180, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770601112713
MOLECULAR RECOGNITION OF ADENOSINE DEAMINASE:
15N NMR STUDIES
Avital Laxer, Hugo E. Gottlieb, and Bilha Fischer
Department of Chemistry,
2
Gonda-Goldschmied Medical Research Center, Bar-Ilan University, Ramat-Gan, Israel
The elucidation of the molecular recognition of adenosine deaminase (ADA), the interpretation
2
of the catalytic mechanism, and the design of novel inhibitors are based mostly on data obtained
for the crystalline state of the enzyme. To obtain evidence for molecular recognition of the
physiologically relevant soluble enzyme, we studied its interactions with the in situ formed inhibitor,
6-OH-purine riboside (HDPR), by 1D-15N- and 2D-(1H-15N)- NMR using the labeled primary
inhibitor [15N4]-PR. We synthesized both [15N4]-PR and an [15N4]-HDPR model, from relatively
inexpensive 15N sources. The [15N4]-HDPR model was used to simulate H-bonding and possible
Zn2+-coordination of HDPR with ADA. We also explored possible ionic interactions between PR
and ADA by 15N-NMR monitored pH-titrations of [15N4]-PR. Finally, we investigated the [15N4]-
PR-ADA 1:1 complex by 2D-(1H-15N) NMR. We found that HDPR recognition determinants in
ADA do not include any ionic-interactions. HDPR N1 H is an H-bond acceptor, and not an
H-bond donor. Despite the proximity of N7 to the Zn2+-ion, no coordination occurs; instead,
N7 is an H-bond acceptor. We found an overall agreement between the crystallographic data
for the crystallized ADA:HDPR complex and the 15N-NMR signals for the corresponding soluble
complex. This finding justifies the use of ADA’s crystallographic data for the design of novel
inhibitors.
Keywords Adenosine deaminase; nucleosides; 15N NMR; Purine riboside
INTRODUCTION
Adenosine deaminase (ADA [Adenosine Aminohydrolase E.C.3.5.4.4])
is a relatively small monomeric enzyme (33 KDa) that catalyzes irreversibly
the hydrolytic deamination of adenosine and 2ꢁ-deoxyadenosine to inosine
and 2ꢁ-deoxy-inosine, respectively.[1,2] Hydrolysis of adenosine and ammo-
nia liberation occurs via a tetrahedral intermediate bearing the amine and
hydroxyl groups at C6.[3] The enzymatic deamination reaction is accel-
erated by about 2 × 1012-times, as compared to deamination in aqueous
solution.[4]
Received 29 June 2006; accepted 6 October 2006.
Address correspondence to Bilha Fischer, Department of Chemistry, Bar-Ilan University, Ramat-Gan
52900, Israel. E-mail: bfischer@mail.biu.ac.il
161

162
A. Laxer et al.
In mammalians, ADA is found in all cells,[5] and a decrease[6] or
increase[7] in ADA levels triggers various pathological conditions. There-
fore, the addition of an efficient and specific inhibitor to ADA is the key to
controlling the activity of endogenous adenosine, involved in various phys-
iological and pathological conditions. Specifically, by elevating adenosine
levels,[8] ADA inhibitors were proposed for the treatment of cerebral and
myocardial ischemia.[9,10]
In addition, inhibition of ADA prevents rapid de-activation of adenosine-
based drugs. For instance, ring-expanded purine nucleosides, including
coformycin and pentostatin, proved synergistic antitumor and/or antiviral
antibiotics which potentiate the effects of other antitumor or antiviral
compounds, through the inhibition of adenosine deaminase.[11] Other
potent ADA inhibitors include deaza-analogues of adenosine and erythro-
9-(2-hydroxy-3-nonyl)adenine (EHNA) analogues.[12–14]
The development of specific and efficient inhibitors requires a thorough
understanding of the molecular recognition determinants of ADA.
The molecular recognition of ADA was investigated by x-ray crystallog-
raphy of ADA-inhibitor complexes.[15,16] For instance, the crystal structure
of the ADA-purine riboside, PR, complex revealed that PR, 1, undergoes
hydration in the catalytic site to form 6-hydroxy-purine-riboside, HDPR,
2a. In this way, the crystallographic data provided significant information
on the transition state of the catalytic reaction, and binding interactions.
HDPR is stabilized in the catalytic site by a network of H-bonds involving
the ribose hydroxyls, and the purine’s N1H, N3, and N7 nitrogen atoms.
For instance, N1H H-bonds to active-site carboxyl (Glu217), while the 6-OH
group interacts with the catalytic site via both H-bonding (Asp295, His238)
and coordination with Zn2+ ion.[16,17]
The crystallographic data provided the basis for the mechanism of
catalysis. [16,18] So far, the elucidation of the molecular recognition of ADA,
the interpretation of the catalytic mechanism, and the design of novel
inhibitors, were based on evidence obtained for the crystalline state of
the enzyme. However, to obtain evidence for molecular recognition of
the soluble enzyme under physiologically relevant conditions, spectroscopic
methods are more suitable than x-ray crystallography.
The application of various spectroscopic techniques is complicated
nevertheless by the multitude data resulting from both the enzyme and
inhibitor partners. Considering both this severe limitation and binding-
modes involving PR nitrogen atoms, we chose 15N NMR for studying the
molecular recognition of PR by soluble ADA.
Specifically, interactions of 1 with the enzyme due to coordination with
metal-ions, protonation, and H-bonds, may be explored by 1D-15N- and 2D-
(1H-15N)- NMR using 15N labeled PR.
Here, we report on the synthesis of [15N4]-PR, 1, from the relatively
inexpensive 15N sources: 15NH4Cl, 15NH4OH, and Na15NO2. Furthermore,

Molecular Recognition of Adenosine Deaminase
163
we report on the synthesis of [15N4]-HDPR model, 2b. This model was
used to simulate H-bonding and possible Zn2+-coordination of HDPR 2a
in the catalytic site, as monitored by 1D-15N-NMR. Likewise, we explored
possible ionic interactions between PR and ADA by 15N-NMR monitored
pH-titrations of [15N4]-PR. Finally, we describe the investigation of [15N4]-
PR-ADA 1:1 complex by 2D-(1H-15N) NMR to elucidate interactions of
HDPR with the catalytic site. This NMR experiment with physiologically
relevant, soluble ADA, complements the molecular recognition picture ob-
tained by crystallography and can confirm or reject hypotheses on binding
interactions deduced for the ADA-HDPR crystalline state.
RESULTS AND DISCUSSION
[15N4]-PR, 1, was the inhibitor of choice for studying the molecular
recognition of ADA by 15N NMR as the affinity of PR to the enzyme is
extremely high (Ki of HDPR is ca. 10−13 M),[19] and it exchanges slowly
(kon is ca. 108 sec−1 mol−1).[20] Such a slow exchange makes it possible
to monitor only the enzyme-bound inhibitor without detecting any free
inhibitor in solution.
Synthesis of [15N4]-PR
Previously, we reported the synthesis of [15N5]-2-thiohexyl-adenine,[21]
9, in five steps from [15N2]-malononitrile and [15N2]-thiourea (Scheme 1).
Each of the starting materials was prepared in two steps utilizing relatively
inexpensive 15N sources: 15NH4Cl, 15NH4OH, and Na15NO2.[21,22]
Here, we utilized labeled adenine analogue 9 as the starting material
for the preparation of [15N4]-PR, 1. The synthetic scheme involved desul-
furization of 9 to obtain [15N5]-adenine followed by coupling to 1-O-acetyl-
2,3,5-tri-O-benzoyl-β-D-ribofuranose, 11, to produce protected and labeled
adenosine analogue, 12 (Scheme 2). Finally, deamination and deprotection
were expected to provide [15N4]-PR, 1. However, desulfurization product,
10, was obtained only in a 25% yield due to its low solubility in organic
solvents and difficulties in removing the product from Ra-Ni.
Alternatively, we coupled [15N4]-2-thiohexyladenine 9 with 11 to pro-
duce nucleoside 13 in 69% yield (Scheme 3). The coupling reaction was
regioselective, occurring at N9 rather than N7. In addition, this reaction
was highly stereoselective, producing the β-isomer as the major product.[23]
The product yield depended on the molar ratio of the base and ribose.
With a 1:1 ratio, the yield was 54%, while with a 1.2:1 base:ribose ratio,
the yield increased to 69%. The excess labeled base can be isolated by
chromatography and recycled.
Desulfurization of 13 was achieved by Ra-Ni in EtOH. Although TLC
indicated the full conversion of the starting material to a single product,

164
A. Laxer et al.
SCHEME 1
SCHEME 2

Molecular Recognition of Adenosine Deaminase
165
SCHEME 3
12, the yield was only 35%, due to the adsorption of the product on the
Ra-Ni, and the limited solubility of 12 in EtOH. Therefore, the reaction was
repeated in DMF at 100◦C. Upon the completion of the reaction, Ra-Ni, was
filtered and washed with warm DMF. In this way, the yield exceeded 60%.
Deamination of 12 was achieved by diazotation using isoamylnitrite.[24,25]
THF in this reaction served as both the solvent and the source of H atoms
reacting with the purine radical formed in the reaction. The protected
PR 14 was obtained in 93% yield. Finally, deprotection of 14 in a mixture
of dioxane-NH4OH-MeOH at 80◦C for 24 hours provided 1 in 90% yield.
This method was found significantly superior to the common deprotection
method in 2 M ethanolic ammonia at 100◦C/24 hours.
Comparison of 15N-chemical shifts of 13 to those of 12 and 14, reveals
a clear downfield shift for all nitrogen atoms in 14 (Table 1). This shift was
most pronounced for N1 and N3. Thus, N1 shifts downfield by 39 ppm due
to removal of the N6-amine and the 2-thioether groups. The lack of these
exocyclic groups, which increase electron density at N1 and N3, results in
deshielding of these nitrogen atoms.
We also compared 15N NMR spectrum of labeled PR in D2O and DMSO.
The large effect of the protic solvent on the nitrogen atoms’ chemical shifts
was clearly observed. N1, N3, and N7 accept H-bonds from water and their
signals shift upfield, compared to their chemical shifts in aprotic solvent
(DMSO), due to reduction of π-bond order on H-bond acceptors.[26] This
effect was not observed for N9 whose lone pair is engaged in the aromatic
system (Table 1).

166
A. Laxer et al.
TABLE 1 15N NMR data for labeled PR and synthetic intermediatesa
Comp no.
Derivative
NH2
N9
N7
N3
N1
13
12
2-Shex-Ad (Bz)
Ad (Bz)
PR (Bz)
PR
−306.0
−305.5
—
—
—
−215.7
−215.1
−214.9
−212.7
−211.1
−142.4
−139.3
−136.3
−147.0
−138.1
−164.5
−154.7
−129.4
−135.5
−129.9
−157.5
−143.5
−118.2
−112.0
−101.0
14
1 (D2O)
1
PR[i]
(DMSO)
aChemical shifts of non-labeled PR.
Upon labeling the PR nitrogen atoms with 15N isotopes, additional 2J N-H
1
couplings appear in the H NMR (Figure 1). Specifically, H6, H2, and H8
appear as follows: d, J = 10; t, J = 14; dd, J = 8, 11 Hz, respectively.
DOES MOLECULAR RECOGNITION OF PR BY ADA INVOLVE
IONIC INTERACTIONS?
Possible protonation of PR by acidic amino-acids in the catalytic site
might affect the binding mode of PR and the catalytic mechanism. Proto-
nation of PR, if any, will result in ionic interactions between the enzyme and
inhibitor partners.
FIGURE 1 1H NMR spectrum of [15N4]-PR, 1: A) full spectrum. b) 2J (N,H) couplings of H-2, H-6, and
H-8.

Molecular Recognition of Adenosine Deaminase
167
TABLE 2 pH-dependence of PR nitrogens 15N NMR chemical shifts
pH
N1
N3
N7
N9
5.63
4.61
3.65
3.04
2.36
1.90
1.39
0.97
−112.1
−112.3
−114.4
−115.8
−129.8
−139.9
−171.1
−185.4
−135.5
−135.5
−135.4
−135.3
−134.5
−133.2
−131.8
−131.1
−147.0
−147.2
−147.0
−147.1
−146.3
−145.3
−144.0
−143.2
−212.7
−212.8
−212.5
−212.3
−212.0
−209.1
−206.7
−205.1
To establish if any ionic interactions are involved in the primary molec-
ular recognition of PR, we determined protonation site and pKa value of PR
by 15N NMR.
In general, ionization constants of nucleos(t)ides are measured by pH-
titration monitored by a variety of spectroscopic methods.[17,27,28] However,
most of these methods do not provide any information on the protonation
site in the nucleobase (e.g., N1 vs. N7). An alternative spectroscopic method
is 15N NMR, which offers unambiguous determination of the protonation
site.[29] Due to the limited solubility of nucleosides, previous 15N NMR
protonation studies of nucleosides have been performed by trifluoroacetic
acid-titrations in DMSO, resulting in no pKa determination.[29,30] Recently,
we demonstrated the use of 15N NMR for the determination of pKa values
of substituted adenine nucleotides.[31]
Here, we first studied the pH-dependent δ of PR nitrogen atoms
(Table 2). Upon acidification of [15N4]-PR solution from pH 5.63 to 0.97,
all nitrogen atoms shifted upfield, however, the most pronounced shift was
observed for N1 with ꢀδ of 73 ppm. The pKa of PR was determined by the
second derivative of the fitted sigmoidal graph of the pH-dependent N1
chemical shift (Figure 2). The PR pKa value was 1.58 ( 0.02). This value
FIGURE 2 pH-titration of [15N4]-PR, 1, monitored by 15N NMR. The sigmoidal graph depicts ꢀδ values
of the [15N4]-PR N1 atom.

168
A. Laxer et al.
is lower as compared to values found in the literature (2.05–2.07).[3,32]
Differences in pKa values may be due to the different determination
methods (UV and 13C NMR vs. 15N NMR), solvent (13C NMR-based pKa was
determined in phosphate buffer), and sample concentrations.
The x-ray crystal structure of the ADA-HDPR complex indicated the
presence of Asp296 in the vicinity of PR’s N7.[15] To test if N7 undergoes any
protonation in the catalytic site, we titrated PR with a strong acid and mon-
itored it by 15N NMR. To a DMSO-d6 solution of PR we added first 1 eq. of
H2SO4 until full protonation of N1 was attained. Then we added HBr (pKa
= −9), but noticed no change in the N7 (or any other nitrogen) chemical
shift. Namely, PR N7 is not basic, unlike the measurable basicity of adeno-
sine N7 (pKa = −1.6).[33] As this experiment was performed in DMSO,
where ionization of acids differs from that in water, we cannot obtain any
quantitative information on PR N7 pKa. However, as N7 is not protonated
by an extremely strong acid as HBr, the basicity of N7 is negligible.
It has been proposed that PR undergoes instantaneous N1-protonation
by Glu217 in the catalytic site.[34,35] However, the low pKa value we deter-
mined for PR N1 implies that the catalytic site must be extremely acidic
for PR protonation to occur. A negative pKa value for Glu is unlikely.
Therefore, we conclude that PR is not initially recognized by ADA via any
ionic-interactions. Furthermore, regarding the C6-hydration mechanism of
PR at the catalytic site, we conclude that N1 is H-bonded to Glu217, rather
than protonated, thus, making C6 more susceptible to hydrolysis.
[15N4]-HDPR MODEL IS REQUIRED FOR 15N NMR STUDIES
OF HDPR:ADA COMPLEX
When PR binds to ADA’s catalytic site a rapid hydration (in less than
4 msec)[20] of the C6-N1 double bond occurs to provide 6-hydroxy-1,6-
dihydropurine riboside, HDPR, 2a. As the addition of labeled PR to ADA
will form labeled HDPR, we had to obtain first 15N NMR data for 2a, for
the assignment of the ADA-bound HDPR 15N chemical shifts in the next
experimental step.
However, HDPR, 2a, is impossible to obtain. Although in the enzyme,
the hydration of PR is quantitative, only traces of HDPR are present in
an aqueous solution, as the equilibrium constant for the hydration is
1.1.10−7.[36] Generally, there is no evidence for hydration of purines and
pyrimidines in an aqueous solution,[37,38] due to the loss of aromaticity
upon hydration. Apparently, HDPR is most stable in ADA’s catalytic site,
due to its resemblance to the substrate’s transition state, and the multitude
of binding interactions with the catalytic site.
SYNTHESIS OF [15N4]-HDPR MODEL
As HDPR is impossible to obtain, we aimed to synthesize a stable
model of HDPR, for example, 15. This model may be obtained from

Molecular Recognition of Adenosine Deaminase
169
PR by alkylation of N1, followed by the addition of a methoxide ion to
C6.
For this purpose, we protected the PR ribose hydroxyls by either benzoyl
or TBDMS groups (14, 16) and have then added MeI. Next, we added
either MeOH/NaOH or MeOH/K2CO3 as methoxide sources. However,
no nucleophilic addition occurred at C6-position in the N1-methylated
PR analogue, 17, under various reaction conditions. Apparently, the silyl
groups reduce the electrophilic nature of 17. Alternatively, we added
MeOH/K2CO3 to N1-methyl per-benzoate PR, 18, in acetonitrile at room
temperature overnight. The addition of MeO− to 18 resulted in the cleavage
of the glycosidic bond and the production of the unstable 1-methyl-6-
hydroxy-dihydropurine, which was detected by MS.
Finally, the desired HDPR model, 2b, was obtained rather unexpectedly,
in a short and facile way, with no need for protection of the ribose hydroxyls
(Scheme 4). Specifically, PR was selectively N1-methylated under mild
conditions by the addition of only 1 eq. MeI in DMSO at room temperature
for 1 week.[36] Separation of the methylated product, 19, on a silica-gel
column resulted in the formation of the desired HDPR model 2b. Although
SCHEME 4

170
A. Laxer et al.
FIGURE 3 Pattern of HDPR model 2b H-1ꢁ signal evaluated at different fields (200 and 600 MHz).
19 had been synthesized before, product 2b was not identified, as 19 was
isolated on cellulose rather than silica gel.[36]
Product 2b was obtained as a mixture of two diastereoisomers, indicated
by the presence of two typical H1ꢁ-signals (two doublets at 5.73 and 5.74
ppm, J = 7 Hz). To ensure that these signals are indeed two doublets and
not a dd signal, we measured the 1H NMR spectrum at different fields (200
1
and 600 MHz) (Figure 3). The different patterns prove that the H signals
result from two diastereoisomeric products.
HDPR model, 2b, obtained easily in one step and in high yield, was
relatively stable in anhydrous solution. Thus, 2b survived in MeOH solution
at −18◦C for several weeks. However, traces of water triggered the decom-
position of 2b. In an aqueous solution, 2b decomposed within minutes.
Assignment of 15N NMR Chemical Shifts of HDPR Model
To assign the chemical shifts of ADA-bound HDPR, we measured the 15
N
NMR spectrum for 2b in a protic medium to mimic H-bond network in the
enzyme. Due to the instability of 2b in water, the spectrum was measured
in CD3OD (Figure 4). The spectrum of 2b was significantly different as
compared to the PR spectrum. Specifically, N1 shifted upfield to ca. 270
ppm due to its sp3 hybridization. Likewise, N3 shifted upfield by ca. 60 ppm
due to the loss of aromaticity.
DOES MOLECULAR RECOGNITION OF HDPR INVOLVE
N7-COORDINATION WITH Zn2+
?
As crystallographic data for the HDPR-ADA complex indicate a relatively
short distance between Zn2+ ion and N7 (3.75 A), HDPR N7 may coordinate
˚
with Zn2+ in soluble ADA. To test this hypothesis, we simulated a possible

Molecular Recognition of Adenosine Deaminase
171
FIGURE 4 15N NMR spectrum of [15N4]-2b in CD3OD at 60.8 MHz.
coordination by the addition of 1 eq. of ZnCl2 to a solution of 2b in CD3OD
and measurement of the resulting 15N NMR chemical shifts. We observed
a significant change of N7 chemical shift by more than 30 ppm, indicating
the coordination of N7 with Zn2+. This coordination reduces the aromatic
character of N7, which turns to be a “pyrrole”-like, trivalent nitrogen. This
observation of upfield shift is consistent with a previous report on an
imidazole-Zn2+ complex.[30,39]
EXPLORING [15N4]-PR RECOGNITION BY ADA BY 2D(1H-15N) NMR
The recognition of PR by ADA may be explored by a high quality 1D-15N
NMR spectrum of a 1:1 PR:ADA complex (exchange is slow). However, for
this purpose, 7–10 mM solutions are required. These high concentrations
result in problems such as low solubility of the protein and high viscosity of
the sample.
To overcome these problems, we chose the 2D-(1H-15N)-INEPT
experiment[40] to study interactions between ADA and PR. In this NMR
experiment the detection of the less sensitive nucleous (15N) is achieved via
the more sensitive nucleus (1H). In this way, we could use less concentrated
samples and reduce measurement time. The INEPT experiment increases
sensitivity 30-fold, and, therefore, a 1–2 mM ADA:PR solution was sufficient
for measurements. A disadvantage of this method is its applicability only to
nitrogens that are coupled to hydrogens. However, polarization transfer is
not limited only to 1J N-H, the value of which is 90 Hz, and information may
be obtained also from long range N-H couplings, when 18 > 3J N-H, 2J N-H
>
5.[41]
2
In purines J N-H are 7–17 Hz, while in pyrimidines they are ca. 17 Hz,
and in imidazole J values are close to 7 Hz.[42]

172
A. Laxer et al.
FIGURE 5 2D-(1H-15N)-HMBC experiment at 1.8 mM [15N4]-PR:ADA complex in 50 mM phosphate
buffer in D2O (pD 7).
To eliminate the large signal of water in the spectrum, we used D2O for
the preparation of phosphate buffer. Although we gain a clearer spectrum,
we lose one bond coupling information resulting from the N1-H labile
proton. Therefore, our experiment of choice was HMBC (suitable for
1H-15N detection through 2–3 bonds) rather than HMQC (suitable for
detection through one bond), as nitrogen atoms in PR are coupled with
the purine hydrogen atoms through two bonds.
The 2D-(1H-15N)-HMBC experiment of ADA:PR 1:1 complex was per-
formed at 1.8 mM concentration in 50 mM phosphate buffer in D2O (pD 7).
The HMBC spectrum showed a cross peak of a nitrogen atom at −268.9 ppm
coupled with a proton at 8.22 ppm (Figure 5). This chemical shift of N
fits perfectly the chemical shift of N1 in the HDPR model, 2b (Figure 4).
3
The proton is H2 and J N-H is 12 Hz. Another cross-peak is observed for a
nitrogen atom that appears at −144.0 ppm coupled with a proton at 8.08
ppm. Here, we also noticed a high correlation with the chemical shift of
N7 and H8 in the HDPR model, 2b in CD3OD. We could not assign the
small cross peak at -260.9 ppm. Cross-peaks corresponding to N3 or N9
were not observed in the HMBC spectrum. In the HMBC experiment, no
2
3
information will be obtained if the peak’s width is larger than the J or J
coupling constant.
CONCLUSIONS
By labeling the inhibitor, rather than the enzyme partner, we were
able to obtain information on the binding interactions of soluble ADA:PR
complex through the 2D-(1H-15N)-HMBC experiment in D2O.

Molecular Recognition of Adenosine Deaminase
173
The HMBC spectrum obtained proved helpful for the understanding of
the molecular recognition of ADA.
A single set of peaks was observed in the HMBC spectrum, thus,
confirming the high affinity of PR to ADA. However, the relatively narrow
width of labeled HDPR N1 and N7 signals in the spectrum, ca. 10 Hz, was
rather unexpected as HDPR is bound to a 33 KDa protein.
The HMBC spectrum provides an additional evidence for the hydration
of PR in the enzyme’s catalytic site by the chemical shift of N1 in the ADA-
bound inhibitor which fits perfectly to the chemical shift of the N1 in HDPR
model, 2b.
Both the H-bonding ADA binding-site and the H-bonding solvent,
MeOH, affect similarly the chemical shift of N1 in [15N4]-HDPR and
[15N4]-HDPR model 2b, respectively. MeOH donates H-bond to N1 in 2b,
which appears at –269 ppm. The chemical shift of ADA-bound HDPR N1H
is identical to this shift. Therefore, we conclude that in ADA’s catalytic
site, HDPR N1 probably experiences an environment similar to that of a
methanolic solution. Furthermore, as in model 2b, there is no hydrogen
atom on N1, but a methyl group, N1 interacts with MeOH only as an
H-bond acceptor. Namely, HDPR N1H in the enzyme catalytic site is also an
H-bond acceptor, and not an H-bond donor. Indications for strong H-bonds
of the HDPR N1H were provided before by Raman[19] and time-dependent
saturation transfer NOE studies.[43] Yet, these studies could not establish the
donor or acceptor role of HDPR N1H.
The chemical shift of N7 in labeled HDPR in the enzyme fits perfectly
with the chemical shift of N7 (an H-bond acceptor) in model 2b in
methanolic solution. This is consistent with the crystallographic evidence
for the interaction of HDPR N7 with Asp296 in ADA.[15]
Based on the null basicity of the PR N7, the low basicity of N1, and
comparison to 2b chemical shifts found here, we conclude that HDPR
recognition determinants in ADA do not include any ionic-interactions with
the neighboring carboxylic acids Asp296 and Glu217, respectively. Likewise,
we did not observe any Zn2+-ion coordination with HDPR N7, unlike N7
in the free 2b. Although Zn2+ ion is within coordination distance,[34] no
significant upfield shift (of ca. 30 ppm) was observed for ADA:HDPR N7
nitrogen. Therefore, we conclude that despite the proximity of N7 to the
Zn2+-ion, no coordination occurs with the metal-ion in the soluble inhibitor-
enzyme complex.
In summary, we conclude that physiologically relevant, soluble, ADA
recognizes HDPR by donating H-bonds to both N1 and N7. We found an
overall agreement between crystallographic data for the ADA:HDPR com-
plex and the 2D-(1H-15N)HMBC experiment for soluble ADA. This finding
justifies the design of novel ADA inhibitors based on ADA’s crystallographic
structure.

174
A. Laxer et al.
EXPERIMENTAL
General
NMR spectra were recorded on a Bruker DPX-300 instrument (300.1,
75.5, and 30.4 MHz for 1H, 13C, 15N, respectively) or on a Bruker DMX-600
instrument (600.1, 150.9, and 60.8 MHz for 1H, 13C, 15N, respectively). The
chemical shifts are reported in ppm relative to tetramethylsilane (TMS) as
an internal standard. 15N NMR spectra were recorded with nitromethane (δ
= 0 ppm) as an external standard. Negative chemical shifts are upfield from
nitromethane. Difficulties in obtaining 15N spectra with good signal to noise
ratios are quite common due to the negative Nuclear Overhauser Effect that
results from the negative magnetogyric ratio of the 15N nucleus, and also
the relative long T1 relaxation times. In order to obtain the necessary data,
spectra were recorded in more than one of the following techniques: proton
coupling, proton decoupling, and inverse gated. Mass spectra were recorded
on AutoSpec-E fision VG high-resolution mass spectrometer. Nucleotides
were characterized by FAB (fast atom bombardment) and high-resolution
FAB using glycerol matrix under FAB negative conditions on AutoSpec-E
fision VG high-resolution mass spectrometer (Micromass, Hertsfordshire,
UK). 15NH4Cl (99% atom 15N) and Na15NO2 (min 99% atom 15N) were
purchased from Isotec Inc. (Miamisburg, OH, USA). (15N2)-Thiourea (96%
atom 15N) and 15NH4OH (98% atom 15N) were purchased from Euriso-
top (Saint Aubin, France), and from Cambridge Isotope Laboratories, Inc.
(Andover, MA, USA) respectively.
[15N4]-Purine Riboside, PR (1): [15N4]-2ꢁ,3ꢁ,5ꢁ-Tri-O-benzoyl-purine ribo-
side 14 (45 mg) was dissolved in dioxane (1.5 mL) and methanol (1 mL) and
added to 28% ammonium hydroxide solution (2 mL) in a sealed pressure
flask equipped with a magnetic stir-bar. The reaction mixture was sealed and
heated at 70–80◦C for 24 hours. The reaction was shown to be complete by
TLC (MeOH:CHCl3; 20:80). The reaction mixture was concentrated under
reduced pressure and the residue was applied to a column of silica gel and
eluted with a gradient of 5–10% MeOH in CHCl3. The product was obtained
1
in quantitative yield as a white solid. H NMR (D2O, 300 MHz) δ: 9.16 (d,
2J (N,H) = 10 Hz, 1H, H-6), 8.96 (t, 2J (N,H) = 14 Hz, 1H, H-2), 8.72 (dd, 2J (N,H)
= 11, 8 Hz, 1H, H-8), 6.23 (d, 2J (H,H) = 5.7 Hz, 1H, H-1ꢁ), 4.87 (t, 2J (H,H)
=
5.3 Hz, 1H, H-2ꢁ), 4.48 (t, 2J (H,H) = 4 Hz, 1H, H-3ꢁ), 4.30 (q, 2J (H,H) = 4 Hz,
1H, H-4ꢁ), 3.94 (dd, 2J (H,H) = 12.8, 3 Hz, 1-H, H-5ꢁ), 3.86 (dd, 2J (H,H) = 12.8,
4 Hz, 1-H, H-5ꢁ). 13C NMR (D2O, 150 MHz) δ: 152.8 (t, 1J (C,N) = 2.8 Hz, C-2),
151.6 (C-4), 149.3 (C-6), 146.6 (d, 1J (C,N) = 11.8 Hz, C-8), 134.8 (C-5), 89.2
(d, 1J (C,N) = 10.8 Hz, C-1ꢁ), 86.4 (C-4ꢁ), 74.6 (C-2ꢁ), 71.2 (C-3ꢁ), 62.1 (C-5ꢁ).
2
15N NMR (D2O, 61 MHz) δ: −112.7 (t, J (N,H) = 11.6, N-1), −136.3 (dd,
2J (H,H) = 14.6, 3 Hz, N-3), −147.6 (N-7), −213.4 (d, 2J (N,H) = 8, N-9). HRMS
(DCI, CH4): calcd for C10H1215N4O4 (M+) 256.0739, found 256.0749.

Molecular Recognition of Adenosine Deaminase
175
1,6-Dihydro-6-hydroxy-1-methyl-9-β-D-ribofuranosyl-9H-purine (2b): A
solution of [15N4]-purine riboside, 1 (42 mg, 0.166 mmol) and methyl
iodide (0.01 mL, 0.166 mmol) in DMSO (0.5 mL) was stirred for one
week at room temperature. The reaction mixture was separated by column
chromatography on silica gel and eluted with CHCl3:MeOH 30:70 to give
the hydration product 2b as an oil (two diastereomers) in a quantitative
1
yield. H NMR (CD3OD, 600 MHz) δ: 7.77 (s, 1 H, H-8), 7.76 (s, 1H,
2
H-8), 7.41 (s, 1H, H-2), 6.24 (s, 1H, H-6), 5.74 (d, J (H,H) = 7 Hz, 1H,
2
2
H-1ꢁ), 5.73 (d, J (H,H) = 7 Hz, 1H, H-1ꢁ), 4.62 (dd, J (H,H) = 7, 5 Hz, 1H,
H-2ꢁ), 4.57 (dd, J (H,H) = 7, 5 Hz, 1H, H-2ꢁ), 4.27–4.25 (m, 1H, H-3ꢁ),
2
4.13 (q, J (H,H) = 2.3 Hz, 1H, H-4ꢁ), 4.12 (q, J (H,H) = 2.3 Hz, 1H, H-4ꢁ),
2
2
3.85–3.80 (m, 1H, H-5ꢁ), 3.68 (dt, J (H,H) = 12.5, 3 Hz, 1H, H-5ꢁ), 3.23
2
(s, 3H, N-CH3), 3.22 (s, 3H, N-CH3). 13C NMR (CD3OD, 150 MHz) δ:
149.81 (C-2), 149.78 (C-2), 137.7 (C-4), 137.6 (C-4), 136.7 (C-8), 136.6
(C-8), 118.4 (C-5), 118.3 (C-5), 91.2 (C-1ꢁ), 91.1 (C-1ꢁ), 88.2 (C-4ꢁ), 88.1
(C-4ꢁ), 86.1 (C-6), 86.0 (C-6), 75.73 (C-2ꢁ), 75.68 (C-2ꢁ), 72.8 (C-3ꢁ), 72.7
(C-4ꢁ), 63.64 (C-5ꢁ), 63.59 (C-5ꢁ), 38.0 (N-CH3), 37.95 (N-CH3). 15N NMR
(CD3OD, 60 MHz) δ: −142.4 (d, 2J (N,H) = 13.4 Hz, N-7), −199.8 (d, 2J (N,H)
2
2
= 12 Hz, N-3), −211.5 (t, J (N,H) = 7.5 Hz, N-9), −268.9 (t, J (N,H)
=
7.5 Hz, N-1). MS: not indicative due to the deglycosylation of 2b in the
mass-spectrometer.
[15N5]-2ꢁ,3ꢁ,5ꢁ-Tri-O-benzoyladenosine (12): A suspension of Raney
nickel and water (50% w/w, about . . . 1 teaspoon) was added to a solution of
[15N5]-2-hexylthio-2ꢁ,3ꢁ,5ꢁ-tri-O-benzoyladenosine 13 (230 mg, 0.328 mmol)
in 30 mL DMF. The heterogeneous solution was heated (100◦C) for 1.5
hours and then filtered and washed with warm DMF. The solvent was
evaporated and co-evaporated with ethanol. The oily residue was purified
by column chromatography and eluted with a gradient of 1–5% MeOH in
CHCl3 to give the product in 60% yield (112 mg). 1H NMR (Acetone-d6, 600
MHz) δ: 8.30 (dd, 2J (N,H) = 11.8, 7.2 Hz, 1H, H-8), 8.20 (t, 2J (N,H) = 15.6 Hz,
1H, H-2), 8.13–7.95(m, 6H, Bz), 7.67–7.58 (m, 3H, Bz), 7.52–7.39 (m, 6H,
1
2
Bz), 6.89 (dd, J (N,H) = 90.5, J (N,N) = 3 Hz, 2H, NH2), 6.69–6.63 (m, 2H,
H-1ꢁ, H-2ꢁ), 6.46 (t, J (N,H) = 5.4 Hz, 1H, H-3ꢁ), 4.98–4.94 (m, 1H, H-4ꢁ),
2
4.92 (dd, 2J (H,H) = 12, 3.5, 1H, H-5ꢁ), 4.78 (dd, 2J (H,H) = 12, 3.5, 1H, H-5ꢁ).
13C NMR (Acetone-d6, 75 MHz) δ: 166.5 (CO), 166.8 (CO), 165.7 (CO),
1
1
157.4 (dm, JC-N = 20.2 Hz, C-6), 154.0 (t, J (C,N) = 2 Hz, C-2), 150.5 (dm,
1J (C,N) = 19.5 Hz, C-4), 141.1 (d, 1J (C,N) = 10.5 Hz, C-8), 134.5, 134.4, 134.1,
130.7, 130.4, 130.1, 129.9, 129.44, 129.38, 88.1 (d, 1J (C,N) = 12 Hz, C-1ꢁ), 80.9
(C-4ꢁ), 74.5 (C-2ꢁ), 72.2 (C-3ꢁ), 64.3 (C-5ꢁ). 15N NMR (Acetone-d6, 61 MHz)
2
2
δ: −139.4 (d, J (N,H) = 14.6 Hz, N-7), −143.6 (N-1), −154.7 (d, J (N,H)
=
17.7 Hz, N-3), −215.1 (d, 2J (N,H) = 5.5 Hz, N-9), −305.4 (td, 1J (N,H) = 91.5,
2J (N,N) = 4.3 Hz, NH2). HRMS (DCI, CH4): calcd for C31H2615N5O7 (MH+)
585.1683, found 585.1698.

176
A. Laxer et al.
[15N5]-2-Hexylthio-2ꢁ,3ꢁ,5ꢁ-tri-O-benzoyladenosine (13): A solution of
[15N5]-2-hexylthioadenine 9 (142 mg, 0.554 mmol) and (NH4)2SO4 (cat-
alytic amount) in HMDS (2 mL) was stirred under reflux for 30 minutes.
The solution was cooled to room temperature and evaporated under high
vacuum. A solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose 11
(155.4 mg, 0.31 mmol) in 1,2-DCE (2 mL) was added to the oily residue of
the silylated base followed by a solution of trimethylsilyl triflate (0.15 mL,
0.83 mmol) in 1,2-DCE (1.85 mL). The mixture was then heated under
reflux for 1 hour, at which time TLC on a silica gel plate (CHCl3:MeOH
9:1) indicated a complete consumption of the sugar and formation of the
product (Rf = 0.69). At this point, the reaction mixture was diluted with
methylene chloride and washed with saturated NaHCO3 solution and brine.
The organic layer was dried with MgSO4, filtered, concentrated, and flash
chromatographed on silica gel by using 3% MeOH in CHCl3 to give the
desired nucleoside 13 in 69% yield (257 mg). 1H NMR (CDCl3, 600 MHz) δ:
8.01–7.19 (m, 6H, Bz), 8.86 (dd, 2J (N,H) = 11.5, 8.5 Hz, 1H, H-8), 7.58–7.53
(m, 3H, Bz), 7.43–7.36 (m, 6H, Bz), 6.40 (ddd, J (H,H) = 5.7, 4, 1.5, 1H,
H-1ꢁ), 6.33 (d, J (H,H) = 4 Hz, 1H, H-2ꢁ), 6.20 (t, J (H,H) = 6.0 Hz, 1H, H-3ꢁ),
1
6.17 (d, J (N,H) = 90.5 Hz, 2H, NH2), 4.85 (dd, J (H,H) = 12, 3.6, 1H, H-5ꢁ),
4.82–4.79 (m, 1H, H-4ꢁ), 4.68 (dd, J (H,H) = 12, 4.5 Hz, 1H, H-5ꢁ), 3.26–3.11
(m, 2H, SCH2), 1.74–1.63 (m, 2H, SCH2CH2), 1.40–1.16 (m, 6H), 0.80 (t,
J (H,H) = 7 Hz, 3H, CH3). 13C NMR (CDCl3, 150 MHz) δ: 166.10 (CO),
1
1
165.93 (CO), 165.06 (d, J (C,N) = 4.4 Hz, C-2), 154.46 (dt, J (C,N) = 20.8
Hz, C-6), 150.10 (dm, 1J (C,N) = 21.4 Hz, C-4), 138.15 (d, 1J (C,N) = 10.9 Hz,
C-8), 133.75, 133.64, 133.39, 129.81, 129.76, 129.59, 129.17, 128.64, 128.56,
128.52, 128.48, 128.46, 117.32 (d, 1J (C,N) = 9.4 Hz, C-5), 87.38 (d, 1J (C,N)
=
12.2 Hz, C-1ꢁ), 80.10 (C-4ꢁ), 74.13 (C-2ꢁ), 71.18 (C-3ꢁ), 63.44 (C-5ꢁ), 31.33,
31.29, 28.77, 28.52, 22.44, 13.95 (CH3). 15N NMR (CD3OD, 61 MHz) δ:
2
−142.7 (N-7), −157.7 (N-1), −164.5 (d, J (N,N) = 3.6 Hz, N-3), −215.7
2
1
2
(d, J (N,H) = 4.9 Hz, N-9), −306.0 (td, J (N,H) = 91.5, J (N,N) = 5.5 Hz,
NH2). HRMS (DCI, CH4): calcd for C37H3715N5O7S (M+) 700.2265, found
700.2222.
[15N4] -2ꢁ,3ꢁ,5ꢁ-Tri-O-benzoylpurine Riboside (14): A solution of [15N5]
-2ꢁ,3ꢁ,5ꢁ-tri-O-benzoyladenosine 12 (78 mg, 0.133 mmol) in dry THF (3.5
mL) was treated with dry iso-amyl nitrite (133 µL, 0.98 mmol) in dry THF
(1 mL). The mixture is immediately purged with nitrogen and then heated
under reflux for 24 hours. An additional aliquot of iso-pentyl nitrite (133
µL) in dry THF (1 mL) was added each day for 2 more days. After 72 hours,
the yellow-orange mixture was evaporated and purified by silica gel column
(eluted with 1% MeOH in CHCl3). The product was obtained as a yellow oil
in 93% yield (73 mg). 1H NMR (CDCl3, 600 MHz) δ: 9.20 (bs, 1H, H-6), 8.91
(t, 2J (N,H) = 14 Hz, H-2), 8.40 (t, 2J (N,H) = 10 Hz, H-8), 8.10–7.90 (m, 6 H,
Bz), 7.62–7.54 (m, 3 H, Bz), 7.47–7.34 (m, 6 H, Bz), 6.53 (d, 2J (H,H) = 4.8 Hz,
1H, H-1ꢁ), 6.41 (t, 2J (H,H) = 4.7 Hz, 1H, H-2ꢁ), 6.26 (t, 2J (H,H) = 5.5 Hz, 1H,

Molecular Recognition of Adenosine Deaminase
177
H-3ꢁ), 4.94 (dd, 2J (H,H) = 12.2, 3.2, 1H, H-5ꢁ), 4.87 (q, 2J (H,H) = 4.2 Hz, 1H,
H-4ꢁ), 4.73 (dd, 2J (H,H) = 12.2, 4.2, 1H, H-5ꢁ). 13C NMR (CDCl3, 150 MHz)
δ: 166.0 (CO), 165.3 (CO), 165.1 (CO), 151.7 (C-4), 151.4 (C-2), 147.2
(C-6), 145.1 (C-8), 134.4 (C-5), 133.9, 133.8, 133.5, 129.8, 129.6, 129.5,
129.2, 128.60, 128.56, 128.5, 128.1, 87.2 (d, 1J (C,N) = 11.4 Hz, C-1ꢁ), 81.0 (C-
4ꢁ), 73.9 (C-2ꢁ), 71.3 (C-3ꢁ), 63.3 (C-5ꢁ). 15N NMR (CDCl3, 61 MHz) δ: −118.2
(N-1), −129.4 (d, 2J (N,H) = 14 Hz, N-3), −136.3 (N-7), −214.9 (N-9). HRMS
(DCI, CH4): calcd for C31H2515N4O7 (MH+) 569.1604, found 569.1646.
1-Methyl-9-(2ꢁ,3ꢁ,5ꢁ-tri-O-tert-butyldimethylsilanyl-β-D-ribofuranosyl)-9H-
Purinium Iodide (17): A solution of 2ꢁ,3ꢁ,5ꢁ-tri-O-(tert-butyldimeth-
ylsilyl)purine riboside 15 (80 mg, 0.134 mmol) and CH3I (0.13 mL,
2.08 mmol) in CH3CN (7 mL) was heated under reflux for 6 hours. After
stirring for an additional 15 hours at room temperature, the solvent was
evaporated under reduced pressure to give the crude ammonium iodide
salt, which was used without further purification in the next synthetic step.
1H NMR (CD3OD, 200 MHz) δ: 9.73 (d, 2J (H,H) = 1.5 Hz, 1H, H-6), 9.48 (d,
2
2J (H,H) = 1.5 Hz, 1H, H-2), 9.44 (s, 1H, H-8), 6.26 (d, J (H,H) = 4 Hz, 1H,
2
H-1ꢁ), 4.78 (t, J (H,H) = 4 Hz, 1H, H-2ꢁ), 4.52–4.45 (m, 4H, H-3ꢁ, N-CH3),
4.24–4.16 (m, 1H, H-4ꢁ), 4.00 (dd, 2J (H,H) = 12.5, 3 Hz, 1H, H-5ꢁ), 3.81 (dd,
2J (H,H) = 12.5, 2.8 Hz, 1H, H-5ꢁ), 0.95 (s, 9H, t-Bu), 0.89 (s, 9H, t-Bu), 0.87
(s, 9H, t-Bu), 0.15 (s, 3H, SiCH3), 0.10 (s, 3H, SiCH3), 0.50 (s, 3H, SiCH3),
0.02 (s, 3H, SiCH3), 0.0 (s, 3H, SiCH3).
Preparation of Adenosine Deaminase Solution for an Enzymatic
Assay and NMR Experiment
A 3.2 M suspension of adenosine deaminase (ADA) from calf intestine
(Roche Diagnostics Corporation, Indianapolis, IN, USA) in (NH4)2SO4
solution was exchanged for 50 mM D2O potassium phosphate buffer, pD 7,
by repeatedly concentrating the sample to less than 0.5 mL and rediluting
to 2 mL with fresh buffer in a distilled-water-rinsed Amicon Centricon-
10 centrifuge concentrator. Six exchange cycles were repeated with the
phosphate buffer in D2O.
ADA reactivity was assayed as follows:[44] 0.1 mL of 1.4 mM adenosine
solution in 0.05 M phosphate buffer, pH 7.4, was added to ice cold 2.88
mL solution of ca. 0.8 units/mL of ADA in 0.05 M phosphate buffer, pH
7.4 (prepared as described above). The decrease in the UV absorbance of
adenosine (at 265 nm) due to its deamination, was measured as a function
of time and the enzyme activity was found to be 146 units/mg.
1H-15N HMBC Experiment of ADA:[15N4]-PR Complex
The NMR sample was prepared as follows: 1.8 mM solution of ADA (0.5
mL, prepared as described above) and [15N4]-PR (0.9 µmol) in 50 mM D2O

178
A. Laxer et al.
potassium phosphate buffer, pD 7 (7.7 µL). 2D-(1H-15N) HMBC spectrum
was recorded on a Bruker DMX-600 instrument (Rheinstetten, Germany).
1
1
(600.1 and 60.8 MHz for H and 15N, respectively). The reference for H
was the residual HDO peak, corrected for the temperature.[45]
The HMBC spectrum was collected using a conventional HMBC pulse
sequence from the Bruker pulse program library. The experiment was run
with a delay time, D6, of 0.04 second, which is optimized for the detection of
12.5 Hz (N-H) coupling constants. The 1H aquisition time was 142.6 msec.
The relaxation delay after each acquisition was set at 2 seconds. The total
experiment time was 16.2 hours. The 2D experiment consisted of 64 time
increments of 1024 data points each. After zero filling in both dimensions
and forward linear prediction in F1 (15N), the final data matrix consisted of
256 × 2048 data points. This resulted in digital resolutions of 1.8 and 47.5
Hz/point for 1H and 15N, respectively.
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