Structure-activity relationships of the antiviral D4T and seven 4´-substituted derivatives using MP2 and DFT methods

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Structural ChemistryComputational and ExperimentalStudies of Chemical and BiologicalSystems ISSN 1040-0400Volume 24Number 3 Struct Chem (2013) 24:967-980DOI 10.1007/s11224-012-0193-x

Structure–activity relationships of theantiviral D4T and seven 4′-substitutedderivatives using MP2 and DFT methods

M. Alcolea Palafox & N. Iza

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ORIGINAL RESEARCH

Structure–activity relationships of the antiviral D4T and seven40-substituted derivatives using MP2 and DFT methods

M. Alcolea Palafox • N. Iza

Received: 20 December 2012 / Accepted: 22 December 2012 / Published online: 15 March 2013

� Springer Science+Business Media New York 2013

Abstract A comprehensive quantum chemical investiga-

tion of the conformational landscapes of the anti-HIV D4T

nucleoside analogue was carried out. Thus, all the possible

stable structures were determined with full optimization of

all the geometrical parameters. The whole conformational

parameters (v, b, c, P, mmax) were analyzed. The hydration

was simulated by explicit water molecules. The most stable

conformer was determined as C1 either in isolated state as

in aqueous solution, b = 63.8�, N-type. Conformer C3

(b = 165.08) is stable with 1–7 water molecules but with a

number larger than eight, C1 is the only stable form in a

close hydrated cluster. However, hydration of the natural

nucleoside thymidine with 13 water molecules stabilizes the

conformer C3 with b ca. 1808 due to the presence of the

C30-OH group. The first phosphorylation step in D4T was

simulated through the interaction with the ATP anion. This

simulation was performed for C1 and C3 conformers in

isolated state, showing that C1 changes to C3 by rotation of

C50-OH group until the value of b ca. 1808. Phosphorylation

of hydrated clusters is only possible with a number of water

molecules below of eight, which permits the C50-OH group

rotation to be accessible for the phosphate group. The

bonding of D4TTP to DNA viral through the reverse

transcriptase enzyme was also simulated. Seven 40-substituted

D4T derivatives were full optimized and analyzed on basis

of the activity reported on TK-1 enzyme and effective

concentration EC50, and several structure–activity rela-

tionships/tendencies were established. The two best cor-

relations correspond to those observed between the TK-1

phosphorylation activity of D4T derivatives and AZT, and

both the calculated exocyclic b angle and dipole moment.

Keywords Stavudine � Zerit � D4T � Structure–activity �Anti-HIV � Phosphorylation

Introduction

Significant progress has been made towards the chemother-

apy (and prophylaxis) of HIV infections [1–5], where

nucleoside analogues have an important role in the current

treatment of cancer and viral infections. Compounds con-

taining an unsaturated ribose ring and with lack of 20- and 30-OH groups, appear as the most effective alternative substrates

of the reverse transcriptase enzyme of the human immuno-

deficiency HIV virus. D4T (20,30-didehydro-20,30-dideoxyt-

hymidine, stavudine or Zerit) (Scheme 1) was one of the first

nucleosides synthesized nearly 40 years ago at the Michigan

Cancer Foundation [6], and Lin and Prusoff [7] at Yale

University discovered the capability of this molecule in

treating HIV/AIDS. Thus, D4T shows exceptional interest. It

was approved by the Food and Drug Administration (FDA)

for clinical use in the infection caused by virus HIV [8, 9]. It

belongs to the Nucleoside Reverse Transcriptase Inhibitors

(NRTIs) antiviral agents that have also wide range of others

biological activity as anti-tumour and antibiotic agents. These

nucleoside analogues are the inactive unphosphorylated form

(prodrugs). Their activation to the triphosphate form by cel-

lular kinases is required for drug potency. Currently, all

Dedicated to Professor Aldo Domenicano on the occasion of his 75th

birthday.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11224-012-0193-x) contains supplementarymaterial, which is available to authorized users.

M. A. Palafox (&) � N. Iza

Departamento de Quımica Fısica I, Facultad de Ciencias

Quımicas, Universidad Complutense, Ciudad Universitaria,

28040 Madrid, Spain

e-mail: [email protected]

123

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DOI 10.1007/s11224-012-0193-x

Author's personal copy

approved NRTIs with the exception of EFdA (40-ethynyl-

2-fluoro-20-deoxyadenosine) lack a 30-OH group. The other

20,30-didehydro-20,30-dideoxynucleosides, containing canon-

ical DNA and RNA bases are also considered as potential RT

inhibitors. It has been shown that there are not conformational

obstacles for incorporation of D4U and D4C into the double

helical A and B forms of DNA [10]. D4C has pronounced

antiretroviral activity [11]. However, D4U, neither its free

[12] nor phosphorylated [13] forms exhibit noticeable bio-

logical activity. This fact can be related to the presence of the

methyl group at the 5-position of the d4T nucleobase and

other modifications at this position of D4U can also transform

the compound into an efficacious RT inhibitors [14]. Intra-

molecular interactions govern conformational properties that

are a decisive factor for binding the active sites of enzymes.

On the other hand, the deoxyribose ring is the major source of

the conformational flexibility of DNA [15]. This is in accord

with the considerable reduction of the conformational

capacity on going from the natural nucleosides to their 20,30-didehydro-20,30-dideoxynucleosides analogues due to the

rigidity of C20=C30double bond in these derivatives.

Several studies have been performed aimed at establishing

the relationship between structure and physicochemical

properties/activity of these drugs. Thus, the anti-HIV-1

activity has been reported that depends upon ribose form

and differences in the ring puckering lead to noticeable

changes in the C50-OH group and in the thymine ring

positions [16]. Recently, relaxed force constants (RFC) and

vibrational root-mean-square (VRMS) deviations have

been used [17] to compare the flexibility of torsional angles

for 20-deoxyribonucleosides (2DRs). It has been found that

torsional angle c is the most rigid one (RFC 15–30 kcal

mol-1) and e, b and the pseudorotation phase angle P are

softer with low RFC values. The glycosidic torsional angle

v is characterized by intermediate values of RFC. The

phenomenon of non-planarity of the amino group in the

amines has also to be considered in nucleobases [18].

From our understanding an analysis of the different con-

formational possibilities for D4T would be interesting, and a

comparison of the results to the nucleoside natural thymidine

(dT) in order to see the facility to change of conformation, the

flexibility of the structure for the interpretation of drug–target

interactions. For this reason different authors have analyzed

the conformers of several nucleosides and nucleosides ana-

logues [19–24]. In previous works we have analyzed the

hydration effect on the two most stables conformers of D4T

[25], and their tautomeric forms [26]. In the present work a

full quantum chemical analysis of the conformational pref-

erences was carried out in an attempt to gain insights into

molecular features responsible for activity. We are also

interested in whether the different H-bondings in D4T make

significant contributions to its conformational behaviour. In

this sense, the importance of intramolecular H-bonds is

showed with 6-azaCyd molecule [27] where the presence of

N6 nitrogen atom leads to the formation of additional, as

compared to Cyd, H-bonds between the nucleoside base and

sugar atoms and allows the molecule to adopt the additional

conformations stabilized by these intramolecular H-bonds.

The variety of conformations made the 6-azaCyd radically

different from the Cyd molecule. Other study on DNA-

related molecules [28] using DFT calculations, Bader’s

quantum theory of atoms in molecules (AIM) and spectro-

scopic vibrational analysis reveals the strongest intramolec-

ular H-bonds with red-shifts over 40 cm-1 in the most cases.

In canonical 20-deoxy- and ribonucleosides, the O50H���N3H

H-bond in ribonucleoside guanosine was found to have the

maximum energy, 8.1 kcal mol-1. In relation to the weaker

H-bonds, MP2 and DFT quantum chemical calculations and

analysis AIM [29] conducts to five types of the CH���Ohydrogen bonds involving bases and sugar residues, and were

detected numerically from 1 to 3 per a conformer: C20H���O50, C10H���O2, C6H���O50, C8H���O50, and C6H���O40. The

energy values of H-bonds are in the range of 2.3–5.6

kcal mol-1, exceeding the kT value (0.62 kcal mol-1). The

Scheme 1 Molecular structure

and definition of the exocyclic

and endocyclic angles in D4T

nucleoside and in seven 40-substituted D4T derivatives

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calculation of the interactions in the dA-T nucleoside pair

gives evidence, that additional to the N6H���O4 and

N1���N3H canonical H-bonds between the bases adenine

and thymine, the third one C2H���O2 is formed, which,

though being rather weak (about 1 kcal mol-1), satisfies

the AIM criteria of H-bonding and may be classified as a

true H-bond. The nucleoside CH���O hydrogen bonds

appeared survive turns of bases against the sugar ring in

rather large ranges of the angle v values, pertinent to cer-

tain conformations. This fact points out to the source of the

DNA characteristic lability necessary for the conforma-

tional adaptation in processes of its functioning.

Another goal of present research is a simulation of the

solid state structure. Several crystallographic studies have

been reported on the polymorphism of D4T [30] yielding to

triclinic [31], monoclinic [32], and orthorhombic [33, 34]

crystals. We have simulated well the dimer form found in

these crystal unit cells.

Efficient phosphorylation depends largely on the spatial

structure of the nucleoside. Thus, an extensive conforma-

tional analysis identifying all minima on the potential

energy surface (PES) can be considered as a first step in the

design of nucleoside analogues as anti-HIV drugs. The first

step of this phosphorylation process was reproduced in the

present manuscript and a proton transfer mechanism in the

isolated state and in the hydrated form was observed.

The last goal was to obtain structure–activity relation-

ships. For this purpose a full optimization of the structural

parameters in seven 40-substituted D4T derivatives was

carried out and the data obtained were compared with the

biological activity reported for these derivatives. As the

formation of monophosphate D4TMP is the first step to

obtain the active metabolite D4TTP, the potential of these

derivatives to be phosphorylated by human TK-1 kinase

was studied. We have simulated the phosphorylation with

an ATP molecule in isolated state and in the presence of

different number of water molecules. In a previous work

[35, 36] we reported that C3 conformation with v =

-122.3�, b = 165�, c = 43.7� is the most adequate to be

phosphorylated at the C50-OH group. Thus, for the first

time structure–activity relations are described here in dT

nucleoside analogues and several conclusions are under-

lined that could be considered for further development of

new anti-HIV drugs.

Calculations

The computations were performed by using the MP2

ab initio method and by using Density Functional methods

(DFT), such as B3LYP and B971 hybrid functionals. These

methods appear implemented in the GAUSSIAN 03

program package [37]. An adequate compromise between

the desired chemical accuracy and the heavy demands put

on computer time and power is provided by DFT methods.

Moreover, these methods were used satisfactory in many

drug design studies [25, 38–40]. The B3LYP method was

selected because several studies have shown that the data

obtained at this level of theory are in good agreement with

those obtained by other more costly computational meth-

ods as MP2 calculations and it predicts the frequencies of

DNA bases better than the HF and MP2 methods [41–45].

Several basis sets were used starting from the 6-31G* to

the 6-311??G(3df,pd), but the 6-31G** basis set was

selected for all the calculations because it represents a

compromise between accuracy and computational cost.

Moreover, these methods and basis sets have been also

used in different nucleosides [10, 19, 46].

Under the TIGHT convergence criterion, the optimum

geometry was determined by minimizing the energy with

respect to all geometrical parameters without imposing

molecular symmetry constraints. The harmonic frequencies

were calculated at the same level of the respective opti-

mization process and by the analytic evaluation of the

second derivative of the energy with respect to the nuclear

displacement. In all the optimized conformers were per-

formed frequency calculations to assess that they corre-

spond to real minimum. All the optimized forms only

showed positive harmonic vibrations (true energy mini-

mum). Relative energies were determined by including

zero-point vibrational energies (ZPE). For the calculation

of ZPE, the wavenumbers were retainted unscaled. Atomic

charges were determined with the Natural NBO [47, 48]

procedure.

All quantum chemical calculations were carried out on

the alpha computer of the Centro de Calculo de la Uni-

versidad Complutense de Madrid, as well as on a HP

Integrity rx2600 server, with 2 Intel� Itanium� 2 proces-

sors at Computational Chemistry Laboratory from Uni-

versidad Nacional de Educacion a Distancia (Lab-QC,

UNED) [49].

The PES was determined in this molecule by rotation of

the torsional angles v (glycosidic bond), c (C40–C50 bond)

and b (C50–O50 bond). In a first study these dihedral angles

simultaneously held fixed at values varying between 0� and

360� in steps of 60� (Table S1 is a resume with the global

minimum) and of 20� in a more detailed second study

(Supplementary Material). During these optimizations all

other geometrical parameters were relaxed. The optimized

points were plotted by the SURFER program [50]. 216

optimized geometries were considered at the first step and

5,832 in the second one.

The ATP molecule was simulated in the completely

deprotonated anionic form with charge -4, and starting

the optimization with X-ray values for their torsional

angles.

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Results and discussion

Definition of the conformational angles

The atomic description of this molecule as well as the most

important endocyclic and exocyclic torsional angles are

defined in Scheme 1 according to the Saenger’s notation

[51]. The different conformations in D4T can be charac-

terized by the following four important structural parame-

ters: (i) the glycosylic torsional angle, v (O40–C10–N1–C2),

which determines the two orientations of the base relative

to the furanose ring, denoted as the anti and syn confor-

mations. (ii) The exocyclic torsional angle c (C30–C40–C50–O50) which describes the orientation of the 50-hydroxyl

group relative to the furanose ring. For minimizing non-

bonded interactions between their substituents this ring is

twisted out-of-plane. (iii) The exocyclic torsional angle b(C40–C50–O50–H50) describes the orientation of the

hydroxyl hydrogen H50 relative to the furanose ring. (iv)

The furanose pucker P defined in the bottom of Table 2,

which indicates north and south orientations.

An important structural characteristic of D4T is the

presence of the C20=C30 double bond, a feature that renders

the sugar ring nearly planar and imparts a high degree of

rigidity to the sugar moiety. However, there are many

conformational possibilities yet for the endocyclic and

exocyclic torsional angles of the molecule.

PES maps in an isolated state

Optimization of the conformers with minimum energies

was carried out, aimed at finding the low-energy confor-

mational states of this molecule. Thus, 25 optimum stable

conformers were determined by rotation of the exocyclic v,

c, and b torsional angles. From all data collected (Sup-

plementary Material) only the 3D conformational map with

the conformers C1–C9 including the global minimum C1

v = -103.68, b = 63.88, c = 60.68 was plotted, Fig. 1. In

this figure the holes correspond to the optimum conform-

ers, which are pointed on the map.

Results by X-ray [31, 32] and molecular mechanics

analysis of conformations [52, 53] have shown an

increased flexibility of the unsaturated thymidine ana-

logues as compared to the saturated compounds. From our

results, it is possible to see that the rotations around C50–O50 bond, b, is less restricted in D4T (energy barrier

height & 2.2 kcal mol-1) than in dT (energy barrier

height = 5.4 kcal mol-1), when v is in anti and c in gg

position. Energy barrier heights are defined here as the

energy difference between the global minimum and the

relevant highest energy point obtained in the PES map. It

can be noted a much lower barrier height in the b direction

than in the c angle. The rotation on the O50–H50 bond is

much easier than on C50–O50. The holes corresponding to

conformers C5, C7 and C6 appear in the conformational

map with v = -103�.

Conformers and energetics

In the isolated state

An extensive conformational study was carried out and the

relative calculated energies of the 25 optimum conformers

were obtained at the B3LYP, B971 and MP2 levels. In the

global minimum a detailed collection of the most impor-

tant conformational parameters, as well as the dipole

moments, is included in Table 1. The conformers were

classified in groups according to the three ranges of

rotation of v: conformers C (v: -119.1� ± 15.5� by MP2),

conformers A (v: -173.6� ± 5.0�) and conformers B

(v: 83.9� ± 18.1�), Fig. 2. The energy criterion by MP2

was followed for the numbering into each group. The C

group contains the most stable conformer at all, C1, and

the majority of more stable conformers. These conformers

C are found in the crystal. The A group conformers, also

found in the crystal, have high planarity. The B group

corresponds to expected non-active biological, syn forms.

Conformers A were difficult to be optimized by the dif-

ferent methods, and in many cases they moved to the

equivalent conformer C.

For each conformer two energy criteria were considered:

Gibbs energy DG, and the electronic energy DE ? ZPE

correction. In the different methods the stability order of the

five most stable conformers by the DE ? ZPE criterion is:

C1 [ C2 [ C3 [ B1 [ C4

by MP2=6-31G�� and B3LYP=6-31G��ð ÞC1 [ C4 [ C3 [ B1 [ C2

by B3LYP=6-311þþG 3df; pfð Þð ÞC1 [ C2 [ C3 [ C4 [ B1

by B971=6-31G��ð Þ

By Gibbs energy criterion is:

C1 [ C4 [ C3 [ C2 [ B1 by B3LYP=6-31G��ð ÞC1 [ C4 [ A1 [ C2 [ C3 by B971=6-31G��ð Þ

The global minimum at all the levels corresponds to the

anti-gg–gg form with respect to v, b and c torsional angles,

-103.6�, 63.8� and 60.6� values by MP2, respectively.

This conformer is denoted as C1 (Fig. 3) and it appears

stabilized by three intramolecular H-bonds/contacts. The

sugar puckering is unsymmetrical twisted, with major O40

atom in endo position and minor C40-exo, 0T4. These

values are close to the expected ones for the natural

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nucleotides and nucleosides in biological systems [51], and

they confirm that the value of v ca. -120� (anti) leads to

the most stable forms.

Figure 3 shows the 12 best structures selected in each

range of the rotation angle v: Six of them correspond to

conformers C (C1 to C6), four to A (A1 to A4), and two to

B (B1, B2). In this figure are also included the values of the

most important H-bonds distances of each conformer. The

computed low-energy B3LYP forms (including the global

minimum) are similar to those determined in dT [54].

The approximate estimated energy barrier height con-

former C1 ? conformer C3, through b direction in the

PES is 3.0 kcal mol-1, while C1 ? C2 is 6.2 kcal mol-1

and C3 ? C2, 3.2 kcal mol-1. The barrier height from

C7 ? C5 is 3.2 kcal mol-1, while C7 ? C6 ? C5 are

0.3 and 0.6 kcal mol-1, respectively. In the c direction the

barrier height is much higher: C1 ? C7 is 6.9 kcal mol-1,

C3 ? C6, 5.5 kcal mol-1, and C2 ? C5, 6.6 kcal mol-1.

Small differences in the structure appear between D4T

and dT: in the intermolecular distances a significant

opening of the structure in dT, with similar O2���O50 and

O40���O50 distances, and slightly longer O4���O50, ca. 0.5 A.

Intramolecular H-bonds

We have considered the classification of the H-bonds

according to Desiraju and Steiner [55]. Thus, five intra-

molecular H-bonds/interaction types may be observed in

the main conformers of D4T [35, 36]: (i) H6���O50, (ii)

H5���O40, (iii) H6���O40, (iv) O2���H10, (v) O2���H50. In

general, the observed strength of H-bonds is as follows:

(v) [ (i) [ (ii) [ (iv) [ (iii).

Conformers C involve the H-bonds/interactions (i), (ii) and

(iv) types while conformers A, the (ii) and (iii), and conformers

B that are achieved by a syn orientation of the thymine, involve

only (v) type. This type of stronger H-bond in the syn forms

gives rise to difficulties in the rotation and orientation of the

hydroxyl group for the phosphorylation process. H-bond (iii)

appears only in conformers A and they are also very weak.

H-bond (v) is the strongest one observed in D4T, and it

appears to give a great stability to the structure of con-

formers B1 and B2. In B1 this H-bond is stronger than in

B2, with a value of O50���H6 by MP2 of 1.888 versus 2.128

A in B2. This strong H-bond is responsible of a high mmax,

as it is observed by MP2.

Fig. 1 Conformational map in

3D and 2D by rotation of b and

c angles for D4T when v =

-123�. The conformation

corresponding to the holes

are indicated

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In the three most stable conformers (C1–C3), H-bond (i) is

weak in accord with the distance 2.452 A for H6���O50 by

MP2 and the C6–H6���O50 angle (126.3�) for C1 conformer

and similar values appear in C2 and C3. This H-bond (i) is

stronger than (ii) and (iv), but weaker than (v). It is noticed

that the anti forms in D4T are characterized by weak or very

weak intramolecular H-bonds and therefore they appear to be

flexible for the different enzymatic process.

Under hydration

It was simulated theoretically by a discrete method (DM)

including explicit water molecules surrounding the com-

pound and compared to the PCM continuum model. In a

hydrated environment, the majority of the optimized con-

formers appear as saddle points by the PCM model. Under

this model the stability order of the different conformers is

drastically changed depending on the level of computation:

C2 [ C3 [ C1 [ C4 [ C5 by B3LYP=6-31G��ð ÞC4 [ C9 [ C3 [ C8 [ C1 by B3LYP=6-31G 2d; pð Þð Þ

As it is expected the conformers with high dipole moment

are favoured in a polar environment with water. Thus, with

the 6-31G** basis set conformers C2 and C3 have the highest

dipole moment 7.59 and 7.64 D, respectively, in accordance

to their highest stability. With DFT methods their values are

in general underestimated, ca. 1 D with the 6-31G** basis set

Table 1 A comparison of the calculated parameters in the most stable conformer C1 of several 40-substituted analogues of D4T: the endocyclic

and exocyclic torsional angles and the pseudorotational angle P are in degrees, the dipole moment l in Debye, and the bond lengths in A

X= v b c ma m0 m1 m2 m3 m4

1 –H -107.6 64.9 62.31 -129.6 -6.97 3.44 1.25 -5.41 7.63

2 –CH3 -106.5 62.6 62.84 -130.8 -7.91 3.75 1.66 -6.30 8.72

3 –CH=CH2 -106.5 62.8 62.90 -130.6 -7.70 3.38 2.04 -6.53 8.71

4 –C:CH -107.4 65.0 62.09 -132.4 -9.66 5.07 1.24 -6.98 10.25

5 –C:C–CH3 -108.3 63.9 62.39 -133.0 -10.33 5.44 1.32 -7.44 10.94

6 –C:C–Cl -108.0 65.3 62.25 -132.7 5.26 5.26 1.25 -7.18 10.57

7 –CH2–

CH=CH2

-106.9 62.2 63.26 -130.8 7.96 3.72 1.75 -6.42 8.83

8 –C:N -107.0 68.0 62.08 -131.6 -8.69 4.68 0.88 -6.13 9.16

P S mmax l O40 charge O50 charge EC50 (lM)b % Activityc

1 80.7 40T 7.7 4.89 -0.595 -0.759 1.3 ± 0.4 106.3 ± 7.7

2 79.2 40T 8.8 5.10 -0.601 -0.757 103.8 ± 9.9

3 76.6 40T 8.8 5.00 -0.594 -0.756 99.5 ± 6.9

4 83.2 0E 10.5 4.67 -0.581 -0.754 0.25 ± 0.14 83.9 ± 6.1

5 83.2 0E 11.2 5.43 -0.583 -0.756 4.0 ± 1.6 74.0 ± 7.9

6 83.3 0E 10.8 4.34 -0.581 -0.754 53.2 ± 9.2

7 78.7 40T 8.9 5.04 -0.599 -0.757 110.8 ± 9.0

8 84.6 0E 9.3 4.02 -0.575 -0.750 7.0 ± 2.6 71.8 ± 8.2

The calculated values correspond to the B3LYP/6-31G** level ? ZPEa m(N1–C10–O40–C40). b EC required to achieve 50 % protection from HIV in MT-2 cells, Ref. [66]. c In the addition of 40-substituted analogues

of D4T to a TK (thymidine kinase) assay, Ref. [66]

Fig. 2 Three types of

conformers determined in D4T

corresponding to the three

ranges of rotation of v with both

b and c * 60�

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and ca. 0.4 D with the 6-311??G(3df,pd) basis set, as

compared to MP2.

Under DM model the first hydration shell of D4T was

simulated using 13 explicit water molecules [25] and 20

water molecules [35, 36] (Fig. 4), in the most stable con-

formers C1, C2 and C3 [56]. C1 and C2 are structurally

similar with b angle 60� and -60�, respectively. Thus,

their hydration clusters are basically the same. Conformer

C3 changes to C1 with addition of more than 8 water

molecules due to water molecules appear between the

thymine and furanose rings, therefore forcing the packing

of the structure and shielding close clusters. In these

clusters with more than eight water molecules, the orien-

tation of the b angle ca. 60� is not appropriate for the

phosphorylation by kinases. The hydration of other con-

formers was also carried out, and they follow similar trend

with a b value ca. 60�, which is neither appropriate for

phosphorylation. Therefore, the cluster with 20 water

Fig. 3 Geometry of the 12 best conformers in decreasing order of stability. The values of the strongest intramolecular H-bond distances, in A,

were determined at B3LY/6-31G**, B3LYP/6-311??G(3df,pd) and MP2/6-31G** (values in parentheses) levels

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molecules of Fig. 4 is the most stable one in a water

environment. We can conclude that in the presence of more

than 8 water molecules per nucleotide, the C1 conformer

will be the only stable one.

By contrast, the hydration of the natural nucleoside

thymidine lead to an orientation of the –O50H group

appropriate for phosphorylation with b ca. 180�, Fig. 5.

This is because the two hydroxyl groups –O50H and –O30Hcan be H-bonded through a water molecule. This fact can

explain the higher phosphorylation of the natural nucleo-

sides than their corresponding 30-deoxynucleosides

analogues.

In the solid state

By X-ray several kinds of D4T crystals have been observed

in the solid state: orthorhombic [33], rhombic [57, 58],

monoclinic [32, 33] and triclinic [31, 33]. In these studies,

as well as in other X-ray studies [59, 60] of related

nucleosides and uridine analogues [61, 62], it is shown that

the glycosyl torsion angle is in anti orientation and their

geometries are stabilized by the formation of self-associ-

ated species. We have well simulated [63] the dimer forms

of the X-ray data with C1 conformers or C1–A2. The

concordance of our calculated values with the experimental

X-ray data, Table 2, confirms the validity of our method-

ology performed on D4T.

Natural NBO atomic charges

It is observed that the largest negative charge in all the

conformers and levels is on the O50 hydroxyl oxygen. Its

value in conformer C1 is similar to that found in dT [25,

26], by MP2 -0.809 e- (where e- is the charge of an

electron). The second atom with large negative charge is

the N3 thymine nitrogen and O2 oxygen. The negative

charge on N3 is larger, ca. 0.2 e-, than on N1 atom. These

features are also in accordance to those calculated in dT

[25] and in AZT [54].

The highest positive charge is on C4 and C2 atoms in

agreement, respectively, to the high negative charge on the

O4 and O2 atoms. C2 has higher positive charge than C4

because of O2 has slight higher negative charge than O4.

The hydrogen atom with the highest positive value corre-

sponds to H50, i.e. it is the most reactive. Closely appears

H3 hydrogen.

First phosphorylation

The first phosphorylation of the nucleosides analogues by

the ATP kinase is a crucial step in the activity of these

prodrugs. The proportion of compound phosphorylated is

in general very small in the majority of the prodrugs. For

understanding this fact, we have simulated first in the

isolated state the interaction of ATP with conformers C1

and C3 of D4T, Fig. 6. It is observed that the interaction of

the phosphate group with the O50-H moiety of conformer

C1 produces a rotation of the b angle to a value ca. 180�,

i.e. conformer C1 changes to C3, and this conformer C3

appears with the b value appropriate for phosphorylation.

The interaction of ATP with the optimum hydrated

clusters of D4T was also simulated. The interaction in the

cluster of C1 conformer with 8 and 12 water molecules is

shown in Fig. 7. It is observed that this interaction of ATP

is not enough strong to rotate the b angle to a value

appropriate for phosphorylation, b ca. 180�. Moreover, the

phosphate groups little affect the values of the b and ctorsional angles. Thus, at this point, it is clear that the

number of water molecules surrounding the nucleoside in

the ATP kinase cavity should be lower than 8.

We study also the interaction of ATP with D4T in the

clusters with lower number than 8 water molecules. For

simplification, in this Fig. 8 is only shown the interaction

of C3 clusters with 6 and 7 water molecules. Similar trend

is observed in the interaction with C1 cluster, because the

phosphate group interaction is strong enough to rotate the

b angle toward the C3 form, which is appropriate for

phosphorylation. Successive steps in the simulation show

Fig. 4 Scheme followed in the

hydration of conformers C1 and

C3 of D4T up to the optimum

cluster with 20 water molecules

reproducing the first hydration

shell. H-bonds D4T-water and

water–water molecules are

denoted by dotted lines

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that the phosphate group with negative charge takes a

hydrogen atom from a neighbour water molecule. Then, the

new OH group formed from this water molecule takes

another H atom from the C50-OH group of D4T. Thus, the

O50 atom is negatively charged and ready to be bonded to a

P atom with positive charge in the ATP. This process

involves one water molecule as observed in the cluster with

6 H2O, or two water molecules in the cluster with 7 H2O.

This feature indicates that the hydrolysis process of ATP in

the phosphorylation, implicates a proton transfer between

ATP and D4T through the neighbour water molecules.

Thus, we can conclude that in the first phosphorylation

by the ATP kinase, the D4T cluster looses some of its water

molecules inside of the enzyme cavity facilitating the

rotation of C50-OH bond to get b ca. 1808, and leading to

interaction with the ATP molecule and the further phos-

phorylation. Therefore, the number of water molecules in

the cavity surrounding the nucleoside is predicted to be

lower than 8.

Bonding of D4TTP to DNA viral

One of the goals of the present paper is the prediction of

new drugs with higher activity than D4T. For this purpose,

we have simulated how the bonding process of D4TTP to

DNA viral is in the cavity of the reverse transcriptase

enzyme, Fig. 9, based on the CHARMm25 force field [64,

65]. It looks clear in this figure that substituents in

Fig. 5 Hydration of the natural

nucleoside thymidine with 13

water molecules stabilizes the

conformer C3 due to C30-OH

group presence. The formation

of two hydrogen bonds between

C50-OH and C30-OH groups and

one water molecule permits a

value of b ca. 180�

Table 2 A comparison of the

calculated dimer forms in D4T

molecule with those reported in

the crystal

The exocyclic torsional angles

and the pseudorotational angle

P are in degrees. The calculated

values correspond to the

B3LYP/6-31G** level ? ZPEam(N1–C10–O40–C40)

Dimer Molecule v c ma P mmax

I A Calculated conformer C1 -108.70 62.04 -129.7 80.86 8.0

X-ray data, orthorhombic -102.1 50.5 -129.0

X-ray data, rhombic -102.0 52.0

B Calculated conformer C1 -107.75 62.80 -129.5 80.69 7.5

X-ray data, orthorhombic -117.2 62.0 -127.6

X-ray data, rhombic -159.9

G/H A Calculated conformer A2 -174.80 44.91 -127.0 90.43 5.3

X-ray data, triclinic -172.6 54.1 -125.6 90.4 4.8

X-ray data, monoclinic -174.1 53.8 -123.1

B Calculated conformer C1 -108.61 62.54 -130.0 81.03 8.3

X-ray data, triclinic -85.1 55.5 -128.8 103.6 6.2

X-ray data, monoclinic -118.0 60.6 -130.5

Fig. 6 First step in the

phosphorylation of conformers

C1 and C3 of D4T by one ATP

molecule in the isolated state. In

this interaction conformer C1

changes to conformer C3 where

C50-OH group with b ca. 180�has the adequate orientation to

loose the proton by the

phosphate moiety

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20-position should be very small for steric interaction with

Residue Y115. In 30-position the substituents can be

something larger than in 20, but not too much. In 40-position

substituents with three carbon atoms are admitted, and

thus, several authors [66, 67] have synthesized D4T

derivatives with different substituents in this position,

Scheme 1. We have studied them in the next section.

Relationships/tendencies observed

The possibility to establish general structure–activity rela-

tionships/tendencies in a series of anti-HIV D4T derivatives

dideoxynucleosides thymidine analogues is another objec-

tive of the present work. Unfortunately, only two papers

report the anti-HIV activity with more than three D4T

derivatives under the same experimental conditions [66, 67].

We have selected Dutschman et al. [66] data due to the bigger

derivative numbers and smaller size of substituents than

those by Argawal et al. [67]. These authors performed

experiments with dT, AZT, D4T and 40-substituted D4T

derivatives, with methyl, vinyl, ethynyl, methylethynyl,

chloroethynyl, allyl and cyano groups, Scheme 1, to evaluate

antiviral effect, cellular toxicity, interaction with TK-1 and

TP enzymes, and pharmacokinetic properties. To start this

study, a full optimization of all compounds was carried

out by B3LYP/6-31G** level ? ZPE to obtain the most

stable conformer. In all cases the global minimum corre-

sponds to the characteristic torsional angles of conformer C1,

as in the former compound D4T, Table 1 and Scheme 1.

Based in all these data, we have related two antiviral

activity parameters, the effective concentration EC50, and

Fig. 7 Interaction of ATP

molecule with the optimum

cluster of D4T in conformer

C1 ? 12H2O and ?8H2O. This

interaction is not enough strong

to rotate the b (C40–C50–O50–H50) exocyclic torsional angle.

H-bonds between D4T-water

and water–water molecules are

denoted by dotted lines

Fig. 8 First step in the phosphorylation of D4T in C3 cluster with 6H2O and 7H2O by one ATP molecule. A proton migration appears from O50-H to the phosphate moiety though one/two water molecules. H-bonds between D4T-water and water–water molecules are denoted by dotted lines

976 Struct Chem (2013) 24:967–980

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the potential of these compounds to be phosphorylated,

named as % activity in a TK-1 kinase assay with [14C]dT

[66]. In this assay, the amount of conversion to [14C]dTMP

with or without addition of analogues in concentration

10-fold-higher than natural nucleoside was compared.

Thus, we have found several interesting structure–activity

relationships/tendencies with our calculated geometric

parameters and dipole moments, some of them are shown

in Fig. 10. The correlation is not so good because the

activity depends on other factors not considered here, but

the tendency is important. Thus, analyzing the different

parameters on these figures was noted the following:

(i) The different substituents on C40 of D4T structure

mainly produce a change in the NBO atomic charge on

O40. An increment in the negative charge on O40 is

linear related to an increase in the negative charge on

O50, a shortening of C40-C50 and a lengthening of C50-O50 and O50-H50. All these changes lead to other effects:

thus an increase in the negative charge on O40 is linear

related to a slight closing of the furanose pucker P and to

an increase in the negative charge on O2 and O4 of the

thymine ring, and as consequence a linear increment in

the dipole moment. Also it is observed a shortening in

the O50���O4 interatomic distance with the increase in

the negative charge on O40. All these features give to the

following conclusion: all the substituents on C40 that

produce an increase in the negative charge on O40,Fig. 10a, and as consequence on O2, O4 and the dipole

moment, increment the activity, Fig. 10e.

(ii) The D4T derivatives with high activity have in

general a low value of the furanose pucker P,

Fig. 10b, and of the exocyclic torsional angle b, but

high torsional angle c in the g? orientation, within

their corresponding ranges, Fig. 10c and d. In these

last figures we have considered important to include

the value of the most used anti-HIV drug AZT, which

follows the tendency observed.

(iii) An increase in the activity in thymidine derivatives is

linearly related to an increment in the dipole

moment, Fig. 10e.

(iv) An increment in the activity appears related to a

lower concentration required to achieve 50 % pro-

tection from HIV in MT-2 cells, EC50, Fig. 10f.

(v) In the few experimental data reported of EC50, a

tendency is suggested between b and c vs. EC50,

Fig. 10g.

(vi) Finally, a lower EC50 value is required in compounds

with a low value of furanose pucker P angle and

dipole moment, Fig. 10h.

We can conclude that the % activity, is related to

high values of the negative charge on O4’ into the

(-0.575 to -0.601) range, dipole moment (4.02-5.43),

c angle (62.088-63.268) and low values of pseudorotation

angle P (76.588-84.608) and b angle (62.248-68.008).Two good correlations are observed for % activity of

D4T derivatives and AZT with b angle and dipole

moment. On the other hand, correlations for antiviral

activity as EC50 from only four derivatives suggest that

Fig. 9 Bonding of D4TTP and

their derivatives under study to

DNA viral through the reverse

transcriptase enzyme

Struct Chem (2013) 24:967–980 977

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is favoured by high b angle values, high % activity, low

P angle and low dipole moment. The c angle has a little

influence on EC50. To confirm the validity of these

relations, we have carried out optimization of 1, 4, 5 and

8 compounds at MP2 level and the results are in

agreement with the tendency observed at B3LYP level.

Additional EC50 data in D4T derivatives will be neces-

sary to confirm these tendencies.

Fig. 10 Structure–activity relationships/tendencies established in seven 40-substituted D4T derivatives

978 Struct Chem (2013) 24:967–980

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Summary and Conclusions

The conformational landscape of the HIV-1 reverse tras-

criptase inhibitor, the nucleoside analogue D4T was explored

using a comprehensive set of modern computational tech-

niques and using four levels of calculation in the isolated

state and two levels in the hydrated form. Comparisons of our

simulated dimer forms with the crystal structure data provide

support for the quality of our quantum chemical calculations.

Thus, the geometries and values presented here appear to

improve the results to date. In the present work the most

important findings are the following:

(1) In general conformers type C are more stable than

conformers type A and B. The calculated five first

optimum conformers are the same by all the methods

and levels. Conformer C1 corresponds to the global

minimum at all the levels of computation, either in

isolated state as in aqueous solution (v = -103.68,b = 63.88, c = 60.68, N-type). In the NBO charges

small differences were observed with dT in AZT.

(2) The first hydration shell was simulated with explicit

water molecules up to 20. The cluster with conformer

C1 was the most stable one. Conformer C3 (v =

-122.38, b = 165.08, c = 43.78), is stable with 1–8

water molecules but with a number of water mole-

cules larger eight, C1 is the only stable form with a

close hydrated cluster.

(3) Hydration of the natural nucleoside thymidine with

13 water molecules stabilizes the conformer C3 with

b ca. 1808 due to the presence of the group C30-OH.

The formation of a molecular pincers between C50-OH and C30-OH groups through two hydrogen bonds

with one water molecule permits a value of b ca. 1808and is stabilized by a four hydrogen bonded water

molecules net.

(4) The first phosphorylation step in D4T was simulated

through the interaction with the ATP anion and for C1

and C3 conformers. The simulation was carried out

under the isolated state consideration as well as under

hydration environment with different amounts of

explicit water molecules. In the isolated state this

interaction is enough strong to change C1 to C3 form

by rotation of C50-OH group until the value of b ca.

1808. This conformer C3 has the value of the b angle

appropriate for the phosphorylation.

(5) Phosphorylation of the D4T hydrated clusters is only

possible with a number of water molecules below of

eight, which permits the C50-OH group rotation to be

accessible for phosphate group. For a successful

phosphorylation, the ATP kinase enzyme must

remove the majority of the water molecules around

the nucleoside and therefore stabilizing conformer

C3, which is the stable form with less than 8 water

molecules.

(6) Hydrolysis process of ATP in the phosphorylation,

implicates a proton transfer between ATP and D4T

through the neighbour water molecules.

(7) Bonding of D4TTP and their derivatives to DNA viral

through the reverse transcriptase enzyme provides

further support for the fact that the biological activity

of 20,30-didehydro-20,30-dideoxy analogues is directly

related to the lack of an OH group at the C30-position.

The study shows also that substituents in 20-position

should be very small for steric interaction with

Residue Y115, substituents in 30-position should not

be large, while substituents in 40-position can be

large.

(8) For the first time structure–activity relationships/

tendencies were established in seven 40-substituted

D4T derivatives and AZT. All the substitutents on C40

in D4T that produce an increase in the negative

charge on O40 (and as consequence on O2, O4 and the

dipole moment) increment the TK-1 % enzymatic

activity.

The first phosphorylation is a crucial step in the activity

of anti-HIV drugs. Good comprehension of the mechanism

that has been proposed for this phosphorylation and all the

parameters investigated here could be very useful for

developing drugs with high anti-HIV activity and low

toxicity. Any information that could shed light on this

problem is important.

Acknowledgments The authors wish to thank to the MCI (Minis-

terio de Ciencia e Innovacion) through CTQ2010-18564 (subprogram

BQU), and to Dra. M. de la Fuente for her great help in the present

work.

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