Scientifica Acta 2, No. 2, 3 – 7 (2008)
Chemistry
Theoretical studies on the reaction of a α-thiono-β-keto-δlactone with sugar-dienophiles
Laura Legnani
Dipartimento di Chimica Organica, Università di Pavia, Viale Taramelli, 10, 27100 Pavia, Italy
[email protected]
Thioether analogues of GM3 -lactone have been proposed as more stable compounds for use as immunogens in the development of a biological therapy against tumor. A synthetic scheme to them is based, in
the key step, on the cycloaddition of a α-thiono-β-keto-δ-lactone, obtainable from D-galactal, with sugar
dienophiles. A theoretical study of this cycloaddition was performed to determine the stereochemical outcome of the reaction and to choose the dienophile that allows to obtain the best GM3 -lactone analogue.
1 Introduction
Oncogenic transformations are often associated with abnormalities in the profiles of cell surface carbohydrates [1]. Although the biological significance of the carbohydrate changes in tumor is not completely
understood, these transformations are supposed to have a specific role on cell-cell communication and on
uncontrolled tumor growth. In the last years, a variety of monoclonal antibodies have been developed to
specifically recognize the carbohydrate epitopes resulting by these faulty glycosylation processes; these
epitopes have been successfully used as markers of tumor progression [2-5] and include carbohydrates
expressed on normal tissues but accumulated in high density on the surface of tumor cells [2].
In particular, several glycosphingolipids have been identified as tumor-associated antigens (TAAs), because of the dramatic changes in transformed cells [1]. Among them there are gangliosides [6], a family
of sialic acid-containing glycosphingolipids present in the outer membrane of living cells and inserted in
the lipid bilayer. The carboxylic group of gangliosides may enter into lactone formation with suitably
placed hydroxyl groups in neighboring saccharide moieties. A large number of carbohydrate structures,
detected in normal tissues, are overexpressed in particular types of tumor cells, such as GM3 -ganglioside
in melanoma cells [7]. The corresponding lactone 1 (Figure 1) has also been found in melanoma. In the
development of a biological therapy of tumor, GM3 -ganglioside and GM3 -lactone 1 have been extensively
investigated as potential vaccines against cancer [8], because of the ability to induce antimelanoma cytotossic T cells (CTL) response.Immunization experiments indicated that the lactone is more immunogenic
than GM3 -ganglioside itself; however its hydrolytic lability makes it less suited as immunogen and hydrolitycally stable and structurally similar lactone analogues are requested. Our attention was focused on the
tricyclic moiety designing analogues in which the lactone function is replaced by a functional group more
stable to hydrolysis and hence potentially more stable than GM3 -lactone under physiological conditions,
for example a thioether derivative such as 2.
A synthetic scheme to the thioether analogues is based, in the key step, on the cycloaddition of diene
3, whose synthesis from D-galactal has been already described [9], with the sugar dienophile 4, obtainable
from D-galactose (Figure 2).
Actually, the synthetic scheme is feasible only if the newly formed stereogenic center, the spiro carbon
atom, is formed with the correct stereochemistry, i.e. the same (S)configuration of the reference compound
1. Thus, before starting the synthetic work, a theoretical study to predict the stereochemical outcome of
the reaction was performed and is here reported. Moreover, in order to determine the rules that govern the
© 2008 Università degli Studi di Pavia
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Scientifica Acta 2, No. 2 (2008)
HO
HO
OH
HO
A cH N
O
H
O
OH
OH
(S )
HO
O
O
O
H
O
OH
HO
A cH N
O
O
H
OH
(S )
HO
OH
O
S
O
H
O C er
HO
OH
1
OH
O
OH
O
O C er
HO
OH
2
Fig. 1: Structure of GM3-lactone 1 and of its thioether analogue 2 (Cer = ceramide).
O
O
O
S
C H 2 O P iv
O
O
O
OBn
+
O
O
O
O
O 3
C H 2 O P iv
(S )
O
O
OBn
O
4
2
O
S
O
Fig. 2: Synthetic scheme for the preparation of 2.
O
O
O
O
O
O
O
O
O
O
5
6
Fig. 3: Structure of the two sugar-dienophiles 5 and 6.
cycloaddition, the study was extended to the two other sugar-dienophiles 5 and 6 obtainable from D-gulose
and D-mannose, respectively (Figure 3).
2 Results
Theoretical calculations on the reaction of the three dienophiles 4-6 with the α-thiono-β-keto-δ-lactone 3
were performed using the Gaussian03 program package through optimization within the DFT approach at
the B3LYP level [10] and the 6-31G(d) basis set. Vibrational frequencies were computed at the same level
of theory in order to define optimized structures as either minima or TS. To allow reasonable computation
times simplified structures were used; in particular: i) the dioxolane substituent at C5 of the pyranose ring
© 2008 Università degli Studi di Pavia
Scientifica Acta 2, No. 2 (2008)
O
5
O
O
O
+
O
S
11
7
O
OCH3
O
CH3
(S )
O
O
OCH3
O
CH3
O
(R )
O
O
S
12
O
O
OCH3
O
O
O
+
O
S
8
CH3
O
O
O
S
13
O
+
OCH3
O
CH3
O
(S )
O
O 10
OCH3
O
(R )
O
O
O
9
O
O
O
OCH3
O
CH3
(S )
O
15
O
S
O
S
14
O
O
CH3
O
+
O
O
O
OCH3
O
CH3
(R )
O
16
O
S
O
Fig. 4: Dienophiles, diene, and cycloadducts considered during the theoretical study.
of 4-6 and the pyvaloyloxymethyl at C5 of lactone 3 were replaced by methyl groups; ii) the isopropylidene methyl groups were replaced by hydrogen atoms; iii) the benzyl group of 3 was replaced by a methyl
group. In Figure 4 are reported these simplified structures 7-10 and the corresponding cycloadducts 11-16.
The reactions of 10 with 7-9 were studied both from the thermodynamical and kinetic point of view. In particular, the relative stability of the pairs of diastereoisomeric adducts 11/12, 13/14, 15/16 was determined
as well as that of the transition states (TS) leading to them. All the degrees of conformational freedom of
the ground state structures 11-16 as well as of the TS were taken into consideration by performing several
optimizations in each case. In the following discussion only the data of the lowest energy conformation of
each structure are reported. In Table 1 are reported the relative reaction energies and activation energies so
determined, whereas in Figure 5 are shown the 3D plots of the most stable conformation of each adduct.
In the cycloaddition of diene 10 with dienophiles 7 and 8, the calculations show that the diastereoisomers
12 and 14, which present the (R) configuration at the spiro carbon atom, are thermodynamically preferred
by about 3 kcal/mol (Table 1). Instead, the cycloaddition of 10 with dienophile 9 shows an opposite
stereoselectivity as the (S)-diastereoisomer 15 is favored over 16 by about 3 kcal/mol.
The same stereochemical outcome is confirmed also under kinetic control. In fact, the TS leading to 12
and 14 resulted favored by 1.36 and 7.47 kcal/mol, respectively, over those leading to 11 and 13 and the
TS leading to 15 by about 10 kcal/mol over that leading to 16. In Figure 6 are shown the 3D plots of the
TS representing the preferred reaction channels of the three cycloadditions.
If the results obtained on the model compounds 7-10 are extended to the corresponding sugar derivatives
3-6, the conclusion that can be drawn is that only 6, the dienophile obtainable from D-mannose, can give,
in the reaction with 3, a cycloadduct with the correct (S) configuration at the spiro carbon atom. This
configuration originates from an attack of 3 to the lower side of the hexacyclic ring. Instead, in the case
of the dienophiles 4 and 5, diene 3 approaches the enolic double bond from the upper side. The drawings
in Figure 7 show that the attack always occurs from the direction opposite to the substituents at C3 and
© 2008 Università degli Studi di Pavia
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Scientifica Acta 2, No. 2 (2008)
11
13
15
12
14
16
Fig. 5: Three-dimensional plots of the preferred conformations of the diastereoisomeric adducts 11/12, 13/14 and
15/16.
Table 1: Relative reaction and activation energies of the cycloadditions of 7-9 with 10.
Compound
11
12
13
14
15
16
TS-12
∆∆E(kcal/mol)
2.77
0.00
3.14
0.00
0.00
3.26
∆∆E… (kcal/mol)
1.36
0.00
7.47
0.00
0.00
9.73
TS-14
TS-15
Fig. 6: Three-dimensional plots of the TS leading to the favorite adducts 12, 14, and 15.
C5. Thus, the configuration of these two centers seems the main factor that governs the stereochemical
outcome of these cycloadditions.
In conclusion, only the dienophile 6 is predicted to be able to give a product with the correct (S)
configuration at the spiro carbon atom. This cycloadduct can be used as the starting point of a synthetic
© 2008 Università degli Studi di Pavia
Scientifica Acta 2, No. 2 (2008)
7
o
o
o
o
o
o
o
o
o
4
o
o
o
5
3
o
4
o
1
2
o
5
6
Fig. 7: Preferred directions of the attack of the diene 3 to the dienophiles 4-6.
sequence to produce an analogue which closely resembles GM3 -lactone in the overall geometric features
[11].
Acknowledgements This work was supported by the Italian Ministero dell’Istruzione, dell’Università e della Ricerca
(COFIN 2006) and by the Universities of Pavia and Florence. Author thanks Professor Lucio Toma and Professor
Franca Marinone Albini for helpful discussions, Professor Cristina Nativi for the fruitful collaboration and CILEA for
the allocation of computer time.
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