- Email: [email protected]
The structure of mono-(3-amino-3-deoxy)-α-cyclodextrin in aqueous solution Molecular dynamics and NMR studies
J o u r n a l of
MOLECULAR STRUCTURE ELSEVIER
Journal of Molecular Structure 442 (1998) 161- 168
The structure of mono-(3-amino-3-deoxy)-a-cyclodextrin in aqueous solution Molecular dynamics and NMR studies Shinji Usui, Tetsuya Tanabe, Hiroshi Ikeda, Akihiko Ueno* Department of Bioengineering, Facul~ of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku. Yokohama 226, Japan
Received 6 June 1997; accepted 6 August 1997
Abstract
The mono-(3-amino-3-deoxy)-c~-cyclodextrin(3-NH 2-~-CD) is a derivative of an c~-cyclodextrin with an amino group at the secondary hydroxyl side. It is known that the glucopyranose residue of cyclodextrins changes to the altrosamine residue when its C3 hydroxyl group is replaced by an amino group. The reported crystal structure of 3-NH2-c~-CD revealed that the altrosamine residue has a skew 3'°B boat conformation. To investigate the conformation of 3-NH2-u-CD in aqueous solution the molecular dynamics (MD) simulation and NMR study were performed in this work. During the MD simulation, the pseudo rotation of the altrosamine residue occurred, i.e. the configuration of altrosamine residue changed from I f 4 to LOB. The dynamic NMR probing indicates that there exists two conformers in aqueous solution. The result suggests that 3-NH2-c~-CD is in an equilibrium between 1C4 and 3'°B forms in aqueous solution. The trajectory MD simulation substantiated that the ring shape of 3-NH2-ot-CD is asymmetrical. © 1998 Elsevier Science B.V. Keywords: Modified cyclodextrin; Conformation; Pseudo rotation; Molecular dynamics; NMR
I. I n t r o d u c t i o n
Cyclodextrins (CDs) are cyclic oligosaccharides which have six or more members o f the o-glucopyranose unit. CDs are torous-shaped hosts with larger and smaller mouths for secondary and primary hydroxyl sides, respectively, with a central hydrophobic cavity. They can form inclusion complexes with various organic compounds in aqueous solution and have been used as model systems o f enzymes and useful vessels for molecules. The cavity size o f CDs is one o f the key factors for their molecular recognition * Fax: 81-45-923-0374. E-maih [email protected]
abilities, and usually not much attention has been paid to their conformations. However, we have shown that conformational features are very important to understand the inclusion phenomena o f CDs, particularly, o f CD derivatives. In fortunate cases, conformations o f CD and CD derivatives in solid state have been determined by X-ray structural analysis [1,2], but the conformations stable in the crystalline state may not be energetically favorable in solution. Under this situation, molecular dynamics (MD) simulation may be a powerful technique for exploring wobbling conformations o f CDs and CD derivatives as well as their complexes particularly in the case when experimental approaches are limited [3-9].
0022-2860/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved PI1 S0022-2 860(97)002 89-5
162
x Ugui et al./Journal t~f Molecular Structure 442 (1998) 161-168
Actually, MD approaches have successively been employed to determine their conformations and mechanisms of molecular recognition. On this basis, the MD conformational analysis may be applied to the new CD systems such as enzymic catalysts [10] or sensors of molecular recognition [11]. With this in mind, we report here the result of MD approach to the key starting compound for secondary site derivatization, mono-(3-amino-3-deoxy)-c~-CD (3-NH2-c~-CD) (Fig. 1). 3-NH2-c~-CD is a derivative of an a-cyclodextrin with an amino group on the secondary hydroxyl side. It is known that the glucopyranose residue of CDs changes to altrosamine residue associated with the replacement of one hydroxyl group by one amino group when it was synthesized from C-2 tosyl c~-CD via 2,3-mannoepoxide [12]. The crystal structure of the 3-NH 2-c~-CD revealed that the altrosamine residue takes the 3'°B boat conformation [13]. To investigate the conformation of the compound in aqueous solution, the molecular dynamics simulation and NMR study were performed. On this basis, asymmetrical shapes of the CD ring, which arises from conversion from glucopyranose to altrose in CD, was envisaged.
2. M e t h o d 2.1. Molecular dynamics simulation
The molecular dynamics simulation was carried out using the DISCOVER/INSIGHTprogram package. The starting coordinate of 3-NH2-c~-CD for the MD simulation was obtained from the reported data of its crystal structure. It was put on the center of the periodic 30,~ cube box. The additional 849 SPC water molecules [14] were inserted into the space not occupied by 3-NH2-t~-CD in the box. The charges for 3-NH2-c~-CD were obtained from parameterized sets with a consistent valence force field (CVFF) [15]. All parameters were used as implemented in a generic parameter set of CVFF. Further minimization was performed on the whole system to stabilize the position of water molecules, constraining only the solute molecule in the starting configuration, until the maximum derivative became less than 0.1 kcal mol -L. This minimized configuration was adopted as
CH20H 5 0°s
0~.~/o....T--_o ,~0,~/
HEk
0
LO~~05. 0
Fig. 1. The structure of 3-NH2-~-CD. Notation of the pyranose residue are also shown. the starting structure of the following MD simulations. Then 300 ps MD simulation was performed after an initial 20 ps equilibration time with constrain to the solute molecule and 12 ps without constrain. A cut-off radius of 10 A was used for Lennard-Jones and electrostatic interactions. After the equilibration, the simulation was performed with periodic boundary conditions and NVT ensemble (constant volume and constant temperature). Every 10 fs the configuration of the solute and solvents were archived, then 30 000 structures were saved from a 300 ps run and were used for data analysis. 2.2. Dynamic N M R probing
The synthesis of 3-NH2-c~-CD was performed according to the procedure reported previously [12]. The H1, H3, and H5 protons were assigned using COSY and TOCSY spectra in the previous report [ 12], while the other protons in the altrosamine residue were not assigned because the peaks of the protons were not separated. Accordingly, the previously assigned three protons were taken into account for investigating the dynamic NMR probing. IH-NMR spectra were recorded on Varian-500S spectrometer at 499.834 MHz. 3-(Trimethylsilyl) propionic acid-d4 sodium salt (TSP, 6 = 0) was used as an external standard. Methanol-d4/D20 (1"2) was used as a
163
S. Usui et al./Journal of Molecular Structure 442 (1998) 161-168
Initial
3'°g form
3~.~
-';:~'--.-,-~f 05
108ps
N N
~'~
3 ~"'--~i~'~
i
~
°$2 f°rm
5
~ ~~'o kJ
110.15 ps
3H2form
110.28 ps 4
•
2il~'>
u.L
1C4form 0
'-"
Fig. 2. The pseudorotation of the altrosamine residue.
solvent to avoid freezing of the solution. The measurements were performed at 5°C initially and then at lower temperatures with an interval of 5°C and the lowest temperature of-15°C.
3. Results and discussions
3.1. Ring pseudorotation of altrosamine residue The primary structural feature driven from the MD trajectory of the 3-NHz-o~-CD is pseudorotation of the altrosamine residue. Fig. 2 shows the ring pseudorotation that occurred in this simulation. Until 108 ps the altrosamine residue maintained the 3'°B form or °$2 form. However, the residue converted to a 3H2 halfchair form at 110.15 ps and then converted to the ~C4 chair form. Furthermore this form was maintained to the last step of MD simulation. The distributions of the dihedral angles in the altrosamine residue are shown in Fig. 3. The distributions
have two peaks in H 1 - C 1 - C 2 - H 2 and H 4 - C 4 - C 5 H5 because the values of dihedral angles were changed by pseudorotation in this simulation. The left and right side peaks in the distribution of H 1 C 1 - C 2 - H 2 indicate that the structures of the altrosamine residue of 3-NH2-o~-CD correspond to ~C4 chair and 3'°B boat forms, respectively. Similarly, in the distribution H 4 - C 4 - C 5 - H 5 has two peaks, indicating that these peaks correspond to 3'°B (left side) and ~C4 (right side) forms. While in H 2 - C 2 - C 3 - H 3 and H 3 - C 3 C4-H4, there is only one peak, indicating that these angles have no variation by pseudorotation. The values of these peaks are summarized in Table 1 together with the simulated and reported NMR coupling constants (JI,2, J2,3, and J4,5) of related protons [12] and the dihedral angles in solid state. Using the coupling constant of 6.6 Hz forJL2 obtained in aqueous solution, the value of the dihedral angle, -152 °, was estimated from the Kurplus equation [16]. As the simulated values for this angle we obtained -168 and -135 °, corresponding to IC 4 and 3'°B
164
S. Usui et al./Journal o[' Molecular Structure 442 (1998) 161-168 1600
1600
H1-C1-C2-H2
1400 1200 1000 e-
--1
o 0
-
H2-C2-C3-H3~'
1400 ~ 1200
-168°(1C4 form)
£
lOOO-
"E 6oo: :3
800 600
-
400
-
0 (,.)
¢/~:~'5°(3'° B form)
200 '~
600"
400:
2°°: t
)
•
Dihedral angle / deg.
I"
I ' 1 " 1 "
I
1600
1400
-
1200
-
1000
-'
"C4H4
'•i
0 600:
•
I"
I"
I"
-72°(1C4 form)
1000
E
800 O (.5 600
-
4OO
200 -
20O " 1 " 1 " 1
I
H4-C4-C5-H5
14001200
t-54
-~ 800 0
"
' 13ihedral angle / deg.
1600
400
t I"
~ ' 1 " 1 " 1 " 1 " 1 " 1 "
Dihedral angle / deg.
-132°(3'°B form)
0
'
•
'
I~ '
i
'
r
'
I
'
i
i
"7"7"7
'
i
~
Dihedral angle / deg.
'
i
~
~
Fig. 3. Distributions of dihedral angles in the altrosamine residue during MD simulation. forms, respectively. Since the v a l u e o f the dihedral angle obtained from the N M R c o u p l i n g constant ( - 1 5 2 °) is the m i d d l e o f the values o f IC4 and 3'°B forms, it was suggested that the c o n f o r m a t i o n o f 3N H z - a - C D resides in an e q u i l i b r i u m b e t w e e n ]C4 and 3'°B forms in aqueous solution.
3.2. D y n a m i c N M R s t u d y
The m e t h o d o f d y n a m i c N M R probing was e m p l o y e d to o b s e r v e the interconversion b e t w e e n IC 4 and 3"°B c o n f o r m a t i o n s o f 3 - N H 2 - a - C D . Generally, the o b s e r v e d N M R spectra o f m o d i f i e d C D s are
Table 1 Dihedral angles and coupling constants of 3-NH2-a-CD Angles
Solid a
Simulation b
HI-C 1-C2-H2
-146.63
H2-C2-C3-H3 H3-C3-C4-H4 H4-C4-C5-H5
-177.18 -51.03 -137.21
-168 (JC4) -135 (3lIB) 179 -54 -72 ( I C 4 ) -132 (3'°B)
J (Hz) c
Calc. d
6.6
-152
10.4 3.8
-154 -39
_
e
a The values of dihedral angles in X-ray structure (Ref. [13]). b The peak values of distribution of dihedral angles in MD simulation/The coupling onstants determined by previous NMR study (Ref. [ 12]).d The calculated values of the dihedral angles using the Kurplus equation (Ref. [ 16]).e The coupling constant was not determined because the H5 proton peaks were not separately observed.
S. Usui et aL/Journal o f Molecular Structure 442 (1998) 161-168
165
(a) ., noo°r°si oo
(b) 3000 2500
i
ooo
DA
I
DB
[t i
g
~
::~,
1500
J
o a_ 1000
i ~
DO ~'
DE
....
iI
500
I
. . . .
4.45
I
. . . .
4.40
I
. . . .
4.35
I
. . . .
4.30
I
. . . .
4.25
3
I
4.20
DF
(ppm)
Fig. 4. IH NMR spectra (500 MHz) of 3-NH2-ct-CD at various temperatures.
the averages of the spectra of the species in various conformational states weighted by the fractions of the conformers. This situation is due to the fact that the interconversion rates are too fast on the NMR time scale. In the present system, as suggested by the above MD simulation, there is a conformational equilibrium between at least two kinds of conformations of 3-NH2-~-CD. If the interconversion rate between two states is slower than 2-~/27rAu, where Au is the difference of their chemical shifts (Hz = s-I), the spectra for two conformations can be observed separately. The NMR spectra of 3-NH2-a-CD are shown in Fig. 4. The 1H resonances for the H5 proton of the altrosamine residue at 4.30 ppm broadened in methanol-d4/D20 (1:2), due to the decreased rate of conformational equilibrium associated with lowering
3.5
4
4.5
5
5.5
Rounded distance /
6
6.5
7
ang.
Fig. 5. (a) The definition of rounded distances of CD ring. The center of each pyranose residue is defined by averaged coordinates of 05, C2, C3, and C5 atoms, and the center of the macrocycle is defined by the averaged coordinate of six 0 4 linker oxygens. (b) The distribution of rounded distances of 3-NH2-ot-CD driven from the MD trajectory.
of temperature to 5°C, and a new small resonance appeared at 0°C. Upon further lowering the temperature, the two resonances designated as A and B in Fig. 4 were completely separated at -15°C. The ~H resonance for the H 1 proton of the altrosamine residue was also decoalesced at -15°C, although these resonances were not clearly separated from the large overlapping HDO resonance around the coalescence temperature. On the other hand, H3 proton did not decoalesce on lowering the temperature. The MD simulation indicated that the dihedral angles around the H3 proton ( H 2 - C 2 - C 3 - H 3 and H 3 - C 3 - C 4 - H 4 )
166
S. Usui et al./Journal of Molecular Structure 442 (1998) 161 168
(b)
D Fig. 6. The snap shot of(a) major and (b) minor conformations of 3-NH 2-¢t-CD driven from the MD trajectory. The altrosamine residue (marked by a capital A) adopts a tC 4 configuration.
are not changed by the conformational change between LOB and IC 4 configurations, and consequently it is reasonable that the environment of the H3 proton is not remarkably affected by pseudorotation between 3'°B and IC4 forms. The decoalesced peaks observed for H1 and H5 indicates that there exists an equilibrium between two conformations of 3-NH2-c~-CD in aqueous solution. The result of the MD simulation may be related to this observation, and the resonances A and B could be assigned to the H5 proton of the IC4 and 3'°B f o r m s , although which of the resonances is which cannot readily be assigned to the two respective forms. The difference in the Gibbs free energy between two conformations was calculated to be about 1.2 kcal mol from the ratio of the resonance area (10:1 for A and B resonances) using the Boltzman equation, and the conformation for the resonance A, which has larger population, is favorable. The band shape analysis g a v e A G t 7 8 = 13.8 kcal mol -l for A to B exchange.
3.3. Distortion of the CD ring The CD ring of native CDs form a symmetrical round shape in aqueous solution because CDs consist
of the same constituent. However, 3-NHz-c~-CD contains an altrosamine residue and consequently it is likely that 3-NH2-c~-CD takes an asymmetrical shape in aqueous solution. We investigated the conformation of the macrocycle of the CD ring by assessing the distance between the center of each pyranose member and the center of the macrocycle (see Fig. 5(a)) during the MD simulation. The distribution of these distances are driven from the trajectory of 300 ps MD simulation which contains both 3'°B and 1C4 configurations for the altrosamine residue. The distribution of these distances are shown in Fig. 5(b). The distances of DB and DE are distributed in the area of about 4 to 5 A with a peak value of distribution of about 4.5 .~. On the other hand, DA, DC, DD, and DF are distributed in the area of about 4.5 to 6 .~ and each peak value spans from 5 to 5.5 ,~, indicating that the distances from the center to the residues B and E are shorter than the others. On this basis, it was suggested that the macrocycle takes an asymmetrical shape which is an ellipse with a shorter distance between B and E glucose residues. Fig. 6(a) shows the snap shot driven from the MD trajectory. The solid structure of 3-NH2-c~-CD indicated a similar shape to that estimated here. The lengths of,
S. Usui et al./Journal o f Molecular Structure 442 (1998) 161-168
(a)
in the solid state, DA, DC, DD, and DF are 4.99, 5.12, 4.94, and 4.99 ,&, respectively, and the lengths of DB and DE are 4.64 and 4.50 A, respectively, displaying a shorter length between B and E residues. The averaged difference between short axes (DB and DE) and long axes (DA, DC, DD, and DF) in the solid structure is 0.44 A. However that difference is about 1 .& in the MD simulation, suggesting that the CD ring of 3-NHz-ot-CD is more distorted in aqueous solution than in the solid.
= mS
167
o to
6" u3
3.4. Dynamic structure of the CD ring .= O nO
=4.0
4.5
5.0
5.5
6.0
6.5
Rounded distance (DA) / ang. o co
(b) c~
o
8c ®
g
o
rr
~a.5
4.0
4.5
5.0
5.5
6.0
Rounded distance (DB) / ang. LO CO
(c)
0
"0
8 n0~
Furthermore, we investigated the dynamic structure of the CD ring of 3-NHz-t~-CD along the time course. Fig. 7 shows the correlations between the distances of (a) DA-DD, (b) DB-DE, and (c) DC-DF, which are the distances of pair residues located at the opposite side beyond the center of the macrocycle (each residue in the pairs is the third residue in the macrocycle from its counterpart). There exists a good correlation for each of the pairs, i.e. when the distance for one residue becomes longer, the distance for its counterpart residue becomes longer. The peak value of the length for DB, which constitutes a short axis of the major conformation of the macrocycle as described previously, is about 4.5 A. However when the length becomes longer than 4.5 ,&, the length of DE also becomes longer. These data allows the existence of minor conformation of the CD ring, whose shape is a circle rather than ellipse. The snap shot of such a conformation is shown in Fig. 6(b). There exists a correlation between DA and DD, and between DC and DF (Fig. 7(a) and 7(c)). When the DA and DC distances become shorter than major length (about 5 to 5.5 ,~) the lengths of DD and DF also become shorter, respectively, indicating that there exists a minor conformation with an ellipse shape of the CD ring whose short axis is different from that of major one. On this basis, it was suggested that the conformation of the CD ring always takes C 2 symmetry which means that, in any case, a pair ofpyranose rings are located at the ends of the axis of the ellipse.
0
~'4.0
4.5
5.0
5.5
6.0
6.5
4. Conclusion
Rounded distance (DC) / ang. Fig. 7. The correlations b e t w e e n r o u n d e d distances for the pair residues driven f r o m the M D trajectory.
The molecular dynamics simulation and dynamic NMR study were applied for 3-NH2-ct-CD to
168
S. Usui et al./Journal of Molecular Structure 442 (1998) 161-168
elucidate its structure in aqueous solution. From the MD simulation and the NMR study it was suggested that there exist two conformers (~C4 and 3'°B) for altrosamine residue in 3-NH2-~-CD. Furthermore, the MD simulation demonstrate that 3-NH2-cc-CD takes an asymmetrical structure of the CD ring with an ellipse shape, in which short axis is directed from B to E pyranose residue (major conformation). In addition there exists a minor conformation with short axis different from the major one. All these simulation data suggested that the ring of 3-NHz-e~CD has C2 symmetry.
Acknowledgements This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports of Japan.
References [ 1] K. B. Lipkowitz, K. Green and J. Yang, Chirality 4 (1992) 205. [2] K. Harata, seimeikougakukougyougijutsukenkyuujo kennkyuuhoukoku 1 (1993) 1. [3] J.E.H. K6hler, W. Saenger and W.F. van Gunsteren, J. Mol. Biol. 203 (1988) 241. [4] M. Prabhakaran, Biochem. Biophys. Res. Commun. 178 (1991) 192. [5] S.P. van Helden, B.P. van Eijick and L.H.M. Janssen, J. Biomol. Struct. and Dynamics 9 (1992) 1269.
[6] J.E.H. K6heler, M. Hohla, M. Richters and W.A. K5nig, Angew. Chem. Int. Engl. 31 (1992) 319. [7] J.E.H. K6heler, M. Hohla, M. Richters and W.A. K6nig Angew, Chem. Ber. 127 (1994) 119. [8] T. Amisaki, T. Fujiwara and S. Kobayashi, J. Mol. Graphic 12 (1994) 297. [9] P.M. Ivanov and C. Jaime, Anales de Qulmica Int. Ed. 92 (1996) 13, [10] (a) Y. Iwakura, K. Uno, F. Toda, S. Onozuka, K. Hattori, M.L. Bender, J. Am. Chem. Soc. 97 (1975) 4432. (b) I. Tabushi, Y. Kuroda, M. Yamada and H. Higashihara, J. Am. Chem. Soc. 107 (1985) 5545. (c) H. Ikeda, R. Kojin, C.-J. Yoon, T. Ikeda and F. Toda, J. Inclusion Phenom. 7 (1989) 117. (d) R. Breslow (Ed.), Supramplecular Chemistry, Kluwer Academic Publishers, Dordrecht, 1991. [ 11 ] (a) T. Kuwabara, A. Nakamura, A. Ueno and F. Toda, J. Phys. Chem. 98 (1994) 6297. (b) H. Ikeda, M. Nakamura, N. lse, N. Oguma, A. Nakamura, T. lkeda, F. Toda and A. Ueno, J. Am. Chem. Soc. 118 (1996) 10980. (c) K. Hamasaki, H. Ikeda, A. Nakamura, A. Ueno and F, Toda, J. Am. Chem. Soc. 115 (1993) 5035. (d) A. Ueno, T. Kuwabara, A. Nakamura and F. Toda, Nature 356 (1992) 136 (f) K. Hamasaki, S. Usui, H. lkeda, T. Ikeda and A. Ueno, Supramol. Chem. 8 (1997) 125. [12] H. Ikeda, Y. Nagano, Y.-Q. Du, T. Ikeda and F. Toda, Tetrahedron Lett. 31 (1990) 5045. [13] K. Harata, Y. Nagano, H. Ikeda, T. Ikeda, A. Ueno and F, Toda, Chem. Commun. 2347 (1996). [14] H,J.C. Berendsen, J.P.M. Postma, W.F. van Gunsteren and J. Hermans, in B. Pullman (Ed.), Intermolecular Force, Reidel Dordrecht, The Netherlands, 1981. [15] DISCOVERUser Guide (version 2.9) Part I, Molecular Simulation Inc., 9685, Scranton Road, San Diego, CA. [16] C,A.G. Haasnoot, F.A.A.M. de leeuw and C. Altona, Tetrahedron 36 (1980) 2783.
MOLECULAR STRUCTURE ELSEVIER
Journal of Molecular Structure 442 (1998) 161- 168
The structure of mono-(3-amino-3-deoxy)-a-cyclodextrin in aqueous solution Molecular dynamics and NMR studies Shinji Usui, Tetsuya Tanabe, Hiroshi Ikeda, Akihiko Ueno* Department of Bioengineering, Facul~ of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku. Yokohama 226, Japan
Received 6 June 1997; accepted 6 August 1997
Abstract
The mono-(3-amino-3-deoxy)-c~-cyclodextrin(3-NH 2-~-CD) is a derivative of an c~-cyclodextrin with an amino group at the secondary hydroxyl side. It is known that the glucopyranose residue of cyclodextrins changes to the altrosamine residue when its C3 hydroxyl group is replaced by an amino group. The reported crystal structure of 3-NH2-c~-CD revealed that the altrosamine residue has a skew 3'°B boat conformation. To investigate the conformation of 3-NH2-u-CD in aqueous solution the molecular dynamics (MD) simulation and NMR study were performed in this work. During the MD simulation, the pseudo rotation of the altrosamine residue occurred, i.e. the configuration of altrosamine residue changed from I f 4 to LOB. The dynamic NMR probing indicates that there exists two conformers in aqueous solution. The result suggests that 3-NH2-c~-CD is in an equilibrium between 1C4 and 3'°B forms in aqueous solution. The trajectory MD simulation substantiated that the ring shape of 3-NH2-ot-CD is asymmetrical. © 1998 Elsevier Science B.V. Keywords: Modified cyclodextrin; Conformation; Pseudo rotation; Molecular dynamics; NMR
I. I n t r o d u c t i o n
Cyclodextrins (CDs) are cyclic oligosaccharides which have six or more members o f the o-glucopyranose unit. CDs are torous-shaped hosts with larger and smaller mouths for secondary and primary hydroxyl sides, respectively, with a central hydrophobic cavity. They can form inclusion complexes with various organic compounds in aqueous solution and have been used as model systems o f enzymes and useful vessels for molecules. The cavity size o f CDs is one o f the key factors for their molecular recognition * Fax: 81-45-923-0374. E-maih [email protected]
abilities, and usually not much attention has been paid to their conformations. However, we have shown that conformational features are very important to understand the inclusion phenomena o f CDs, particularly, o f CD derivatives. In fortunate cases, conformations o f CD and CD derivatives in solid state have been determined by X-ray structural analysis [1,2], but the conformations stable in the crystalline state may not be energetically favorable in solution. Under this situation, molecular dynamics (MD) simulation may be a powerful technique for exploring wobbling conformations o f CDs and CD derivatives as well as their complexes particularly in the case when experimental approaches are limited [3-9].
0022-2860/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved PI1 S0022-2 860(97)002 89-5
162
x Ugui et al./Journal t~f Molecular Structure 442 (1998) 161-168
Actually, MD approaches have successively been employed to determine their conformations and mechanisms of molecular recognition. On this basis, the MD conformational analysis may be applied to the new CD systems such as enzymic catalysts [10] or sensors of molecular recognition [11]. With this in mind, we report here the result of MD approach to the key starting compound for secondary site derivatization, mono-(3-amino-3-deoxy)-c~-CD (3-NH2-c~-CD) (Fig. 1). 3-NH2-c~-CD is a derivative of an a-cyclodextrin with an amino group on the secondary hydroxyl side. It is known that the glucopyranose residue of CDs changes to altrosamine residue associated with the replacement of one hydroxyl group by one amino group when it was synthesized from C-2 tosyl c~-CD via 2,3-mannoepoxide [12]. The crystal structure of the 3-NH 2-c~-CD revealed that the altrosamine residue takes the 3'°B boat conformation [13]. To investigate the conformation of the compound in aqueous solution, the molecular dynamics simulation and NMR study were performed. On this basis, asymmetrical shapes of the CD ring, which arises from conversion from glucopyranose to altrose in CD, was envisaged.
2. M e t h o d 2.1. Molecular dynamics simulation
The molecular dynamics simulation was carried out using the DISCOVER/INSIGHTprogram package. The starting coordinate of 3-NH2-c~-CD for the MD simulation was obtained from the reported data of its crystal structure. It was put on the center of the periodic 30,~ cube box. The additional 849 SPC water molecules [14] were inserted into the space not occupied by 3-NH2-t~-CD in the box. The charges for 3-NH2-c~-CD were obtained from parameterized sets with a consistent valence force field (CVFF) [15]. All parameters were used as implemented in a generic parameter set of CVFF. Further minimization was performed on the whole system to stabilize the position of water molecules, constraining only the solute molecule in the starting configuration, until the maximum derivative became less than 0.1 kcal mol -L. This minimized configuration was adopted as
CH20H 5 0°s
0~.~/o....T--_o ,~0,~/
HEk
0
LO~~05. 0
Fig. 1. The structure of 3-NH2-~-CD. Notation of the pyranose residue are also shown. the starting structure of the following MD simulations. Then 300 ps MD simulation was performed after an initial 20 ps equilibration time with constrain to the solute molecule and 12 ps without constrain. A cut-off radius of 10 A was used for Lennard-Jones and electrostatic interactions. After the equilibration, the simulation was performed with periodic boundary conditions and NVT ensemble (constant volume and constant temperature). Every 10 fs the configuration of the solute and solvents were archived, then 30 000 structures were saved from a 300 ps run and were used for data analysis. 2.2. Dynamic N M R probing
The synthesis of 3-NH2-c~-CD was performed according to the procedure reported previously [12]. The H1, H3, and H5 protons were assigned using COSY and TOCSY spectra in the previous report [ 12], while the other protons in the altrosamine residue were not assigned because the peaks of the protons were not separated. Accordingly, the previously assigned three protons were taken into account for investigating the dynamic NMR probing. IH-NMR spectra were recorded on Varian-500S spectrometer at 499.834 MHz. 3-(Trimethylsilyl) propionic acid-d4 sodium salt (TSP, 6 = 0) was used as an external standard. Methanol-d4/D20 (1"2) was used as a
163
S. Usui et al./Journal of Molecular Structure 442 (1998) 161-168
Initial
3'°g form
3~.~
-';:~'--.-,-~f 05
108ps
N N
~'~
3 ~"'--~i~'~
i
~
°$2 f°rm
5
~ ~~'o kJ
110.15 ps
3H2form
110.28 ps 4
•
2il~'>
u.L
1C4form 0
'-"
Fig. 2. The pseudorotation of the altrosamine residue.
solvent to avoid freezing of the solution. The measurements were performed at 5°C initially and then at lower temperatures with an interval of 5°C and the lowest temperature of-15°C.
3. Results and discussions
3.1. Ring pseudorotation of altrosamine residue The primary structural feature driven from the MD trajectory of the 3-NHz-o~-CD is pseudorotation of the altrosamine residue. Fig. 2 shows the ring pseudorotation that occurred in this simulation. Until 108 ps the altrosamine residue maintained the 3'°B form or °$2 form. However, the residue converted to a 3H2 halfchair form at 110.15 ps and then converted to the ~C4 chair form. Furthermore this form was maintained to the last step of MD simulation. The distributions of the dihedral angles in the altrosamine residue are shown in Fig. 3. The distributions
have two peaks in H 1 - C 1 - C 2 - H 2 and H 4 - C 4 - C 5 H5 because the values of dihedral angles were changed by pseudorotation in this simulation. The left and right side peaks in the distribution of H 1 C 1 - C 2 - H 2 indicate that the structures of the altrosamine residue of 3-NH2-o~-CD correspond to ~C4 chair and 3'°B boat forms, respectively. Similarly, in the distribution H 4 - C 4 - C 5 - H 5 has two peaks, indicating that these peaks correspond to 3'°B (left side) and ~C4 (right side) forms. While in H 2 - C 2 - C 3 - H 3 and H 3 - C 3 C4-H4, there is only one peak, indicating that these angles have no variation by pseudorotation. The values of these peaks are summarized in Table 1 together with the simulated and reported NMR coupling constants (JI,2, J2,3, and J4,5) of related protons [12] and the dihedral angles in solid state. Using the coupling constant of 6.6 Hz forJL2 obtained in aqueous solution, the value of the dihedral angle, -152 °, was estimated from the Kurplus equation [16]. As the simulated values for this angle we obtained -168 and -135 °, corresponding to IC 4 and 3'°B
164
S. Usui et al./Journal o[' Molecular Structure 442 (1998) 161-168 1600
1600
H1-C1-C2-H2
1400 1200 1000 e-
--1
o 0
-
H2-C2-C3-H3~'
1400 ~ 1200
-168°(1C4 form)
£
lOOO-
"E 6oo: :3
800 600
-
400
-
0 (,.)
¢/~:~'5°(3'° B form)
200 '~
600"
400:
2°°: t
)
•
Dihedral angle / deg.
I"
I ' 1 " 1 "
I
1600
1400
-
1200
-
1000
-'
"C4H4
'•i
0 600:
•
I"
I"
I"
-72°(1C4 form)
1000
E
800 O (.5 600
-
4OO
200 -
20O " 1 " 1 " 1
I
H4-C4-C5-H5
14001200
t-54
-~ 800 0
"
' 13ihedral angle / deg.
1600
400
t I"
~ ' 1 " 1 " 1 " 1 " 1 " 1 "
Dihedral angle / deg.
-132°(3'°B form)
0
'
•
'
I~ '
i
'
r
'
I
'
i
i
"7"7"7
'
i
~
Dihedral angle / deg.
'
i
~
~
Fig. 3. Distributions of dihedral angles in the altrosamine residue during MD simulation. forms, respectively. Since the v a l u e o f the dihedral angle obtained from the N M R c o u p l i n g constant ( - 1 5 2 °) is the m i d d l e o f the values o f IC4 and 3'°B forms, it was suggested that the c o n f o r m a t i o n o f 3N H z - a - C D resides in an e q u i l i b r i u m b e t w e e n ]C4 and 3'°B forms in aqueous solution.
3.2. D y n a m i c N M R s t u d y
The m e t h o d o f d y n a m i c N M R probing was e m p l o y e d to o b s e r v e the interconversion b e t w e e n IC 4 and 3"°B c o n f o r m a t i o n s o f 3 - N H 2 - a - C D . Generally, the o b s e r v e d N M R spectra o f m o d i f i e d C D s are
Table 1 Dihedral angles and coupling constants of 3-NH2-a-CD Angles
Solid a
Simulation b
HI-C 1-C2-H2
-146.63
H2-C2-C3-H3 H3-C3-C4-H4 H4-C4-C5-H5
-177.18 -51.03 -137.21
-168 (JC4) -135 (3lIB) 179 -54 -72 ( I C 4 ) -132 (3'°B)
J (Hz) c
Calc. d
6.6
-152
10.4 3.8
-154 -39
_
e
a The values of dihedral angles in X-ray structure (Ref. [13]). b The peak values of distribution of dihedral angles in MD simulation/The coupling onstants determined by previous NMR study (Ref. [ 12]).d The calculated values of the dihedral angles using the Kurplus equation (Ref. [ 16]).e The coupling constant was not determined because the H5 proton peaks were not separately observed.
S. Usui et aL/Journal o f Molecular Structure 442 (1998) 161-168
165
(a) ., noo°r°si oo
(b) 3000 2500
i
ooo
DA
I
DB
[t i
g
~
::~,
1500
J
o a_ 1000
i ~
DO ~'
DE
....
iI
500
I
. . . .
4.45
I
. . . .
4.40
I
. . . .
4.35
I
. . . .
4.30
I
. . . .
4.25
3
I
4.20
DF
(ppm)
Fig. 4. IH NMR spectra (500 MHz) of 3-NH2-ct-CD at various temperatures.
the averages of the spectra of the species in various conformational states weighted by the fractions of the conformers. This situation is due to the fact that the interconversion rates are too fast on the NMR time scale. In the present system, as suggested by the above MD simulation, there is a conformational equilibrium between at least two kinds of conformations of 3-NH2-~-CD. If the interconversion rate between two states is slower than 2-~/27rAu, where Au is the difference of their chemical shifts (Hz = s-I), the spectra for two conformations can be observed separately. The NMR spectra of 3-NH2-a-CD are shown in Fig. 4. The 1H resonances for the H5 proton of the altrosamine residue at 4.30 ppm broadened in methanol-d4/D20 (1:2), due to the decreased rate of conformational equilibrium associated with lowering
3.5
4
4.5
5
5.5
Rounded distance /
6
6.5
7
ang.
Fig. 5. (a) The definition of rounded distances of CD ring. The center of each pyranose residue is defined by averaged coordinates of 05, C2, C3, and C5 atoms, and the center of the macrocycle is defined by the averaged coordinate of six 0 4 linker oxygens. (b) The distribution of rounded distances of 3-NH2-ot-CD driven from the MD trajectory.
of temperature to 5°C, and a new small resonance appeared at 0°C. Upon further lowering the temperature, the two resonances designated as A and B in Fig. 4 were completely separated at -15°C. The ~H resonance for the H 1 proton of the altrosamine residue was also decoalesced at -15°C, although these resonances were not clearly separated from the large overlapping HDO resonance around the coalescence temperature. On the other hand, H3 proton did not decoalesce on lowering the temperature. The MD simulation indicated that the dihedral angles around the H3 proton ( H 2 - C 2 - C 3 - H 3 and H 3 - C 3 - C 4 - H 4 )
166
S. Usui et al./Journal of Molecular Structure 442 (1998) 161 168
(b)
D Fig. 6. The snap shot of(a) major and (b) minor conformations of 3-NH 2-¢t-CD driven from the MD trajectory. The altrosamine residue (marked by a capital A) adopts a tC 4 configuration.
are not changed by the conformational change between LOB and IC 4 configurations, and consequently it is reasonable that the environment of the H3 proton is not remarkably affected by pseudorotation between 3'°B and IC4 forms. The decoalesced peaks observed for H1 and H5 indicates that there exists an equilibrium between two conformations of 3-NH2-c~-CD in aqueous solution. The result of the MD simulation may be related to this observation, and the resonances A and B could be assigned to the H5 proton of the IC4 and 3'°B f o r m s , although which of the resonances is which cannot readily be assigned to the two respective forms. The difference in the Gibbs free energy between two conformations was calculated to be about 1.2 kcal mol from the ratio of the resonance area (10:1 for A and B resonances) using the Boltzman equation, and the conformation for the resonance A, which has larger population, is favorable. The band shape analysis g a v e A G t 7 8 = 13.8 kcal mol -l for A to B exchange.
3.3. Distortion of the CD ring The CD ring of native CDs form a symmetrical round shape in aqueous solution because CDs consist
of the same constituent. However, 3-NHz-c~-CD contains an altrosamine residue and consequently it is likely that 3-NH2-c~-CD takes an asymmetrical shape in aqueous solution. We investigated the conformation of the macrocycle of the CD ring by assessing the distance between the center of each pyranose member and the center of the macrocycle (see Fig. 5(a)) during the MD simulation. The distribution of these distances are driven from the trajectory of 300 ps MD simulation which contains both 3'°B and 1C4 configurations for the altrosamine residue. The distribution of these distances are shown in Fig. 5(b). The distances of DB and DE are distributed in the area of about 4 to 5 A with a peak value of distribution of about 4.5 .~. On the other hand, DA, DC, DD, and DF are distributed in the area of about 4.5 to 6 .~ and each peak value spans from 5 to 5.5 ,~, indicating that the distances from the center to the residues B and E are shorter than the others. On this basis, it was suggested that the macrocycle takes an asymmetrical shape which is an ellipse with a shorter distance between B and E glucose residues. Fig. 6(a) shows the snap shot driven from the MD trajectory. The solid structure of 3-NH2-c~-CD indicated a similar shape to that estimated here. The lengths of,
S. Usui et al./Journal o f Molecular Structure 442 (1998) 161-168
(a)
in the solid state, DA, DC, DD, and DF are 4.99, 5.12, 4.94, and 4.99 ,&, respectively, and the lengths of DB and DE are 4.64 and 4.50 A, respectively, displaying a shorter length between B and E residues. The averaged difference between short axes (DB and DE) and long axes (DA, DC, DD, and DF) in the solid structure is 0.44 A. However that difference is about 1 .& in the MD simulation, suggesting that the CD ring of 3-NHz-ot-CD is more distorted in aqueous solution than in the solid.
= mS
167
o to
6" u3
3.4. Dynamic structure of the CD ring .= O nO
=4.0
4.5
5.0
5.5
6.0
6.5
Rounded distance (DA) / ang. o co
(b) c~
o
8c ®
g
o
rr
~a.5
4.0
4.5
5.0
5.5
6.0
Rounded distance (DB) / ang. LO CO
(c)
0
"0
8 n0~
Furthermore, we investigated the dynamic structure of the CD ring of 3-NHz-t~-CD along the time course. Fig. 7 shows the correlations between the distances of (a) DA-DD, (b) DB-DE, and (c) DC-DF, which are the distances of pair residues located at the opposite side beyond the center of the macrocycle (each residue in the pairs is the third residue in the macrocycle from its counterpart). There exists a good correlation for each of the pairs, i.e. when the distance for one residue becomes longer, the distance for its counterpart residue becomes longer. The peak value of the length for DB, which constitutes a short axis of the major conformation of the macrocycle as described previously, is about 4.5 A. However when the length becomes longer than 4.5 ,&, the length of DE also becomes longer. These data allows the existence of minor conformation of the CD ring, whose shape is a circle rather than ellipse. The snap shot of such a conformation is shown in Fig. 6(b). There exists a correlation between DA and DD, and between DC and DF (Fig. 7(a) and 7(c)). When the DA and DC distances become shorter than major length (about 5 to 5.5 ,~) the lengths of DD and DF also become shorter, respectively, indicating that there exists a minor conformation with an ellipse shape of the CD ring whose short axis is different from that of major one. On this basis, it was suggested that the conformation of the CD ring always takes C 2 symmetry which means that, in any case, a pair ofpyranose rings are located at the ends of the axis of the ellipse.
0
~'4.0
4.5
5.0
5.5
6.0
6.5
4. Conclusion
Rounded distance (DC) / ang. Fig. 7. The correlations b e t w e e n r o u n d e d distances for the pair residues driven f r o m the M D trajectory.
The molecular dynamics simulation and dynamic NMR study were applied for 3-NH2-ct-CD to
168
S. Usui et al./Journal of Molecular Structure 442 (1998) 161-168
elucidate its structure in aqueous solution. From the MD simulation and the NMR study it was suggested that there exist two conformers (~C4 and 3'°B) for altrosamine residue in 3-NH2-~-CD. Furthermore, the MD simulation demonstrate that 3-NH2-cc-CD takes an asymmetrical structure of the CD ring with an ellipse shape, in which short axis is directed from B to E pyranose residue (major conformation). In addition there exists a minor conformation with short axis different from the major one. All these simulation data suggested that the ring of 3-NHz-e~CD has C2 symmetry.
Acknowledgements This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports of Japan.
References [ 1] K. B. Lipkowitz, K. Green and J. Yang, Chirality 4 (1992) 205. [2] K. Harata, seimeikougakukougyougijutsukenkyuujo kennkyuuhoukoku 1 (1993) 1. [3] J.E.H. K6hler, W. Saenger and W.F. van Gunsteren, J. Mol. Biol. 203 (1988) 241. [4] M. Prabhakaran, Biochem. Biophys. Res. Commun. 178 (1991) 192. [5] S.P. van Helden, B.P. van Eijick and L.H.M. Janssen, J. Biomol. Struct. and Dynamics 9 (1992) 1269.
[6] J.E.H. K6heler, M. Hohla, M. Richters and W.A. K5nig, Angew. Chem. Int. Engl. 31 (1992) 319. [7] J.E.H. K6heler, M. Hohla, M. Richters and W.A. K6nig Angew, Chem. Ber. 127 (1994) 119. [8] T. Amisaki, T. Fujiwara and S. Kobayashi, J. Mol. Graphic 12 (1994) 297. [9] P.M. Ivanov and C. Jaime, Anales de Qulmica Int. Ed. 92 (1996) 13, [10] (a) Y. Iwakura, K. Uno, F. Toda, S. Onozuka, K. Hattori, M.L. Bender, J. Am. Chem. Soc. 97 (1975) 4432. (b) I. Tabushi, Y. Kuroda, M. Yamada and H. Higashihara, J. Am. Chem. Soc. 107 (1985) 5545. (c) H. Ikeda, R. Kojin, C.-J. Yoon, T. Ikeda and F. Toda, J. Inclusion Phenom. 7 (1989) 117. (d) R. Breslow (Ed.), Supramplecular Chemistry, Kluwer Academic Publishers, Dordrecht, 1991. [ 11 ] (a) T. Kuwabara, A. Nakamura, A. Ueno and F. Toda, J. Phys. Chem. 98 (1994) 6297. (b) H. Ikeda, M. Nakamura, N. lse, N. Oguma, A. Nakamura, T. lkeda, F. Toda and A. Ueno, J. Am. Chem. Soc. 118 (1996) 10980. (c) K. Hamasaki, H. Ikeda, A. Nakamura, A. Ueno and F, Toda, J. Am. Chem. Soc. 115 (1993) 5035. (d) A. Ueno, T. Kuwabara, A. Nakamura and F. Toda, Nature 356 (1992) 136 (f) K. Hamasaki, S. Usui, H. lkeda, T. Ikeda and A. Ueno, Supramol. Chem. 8 (1997) 125. [12] H. Ikeda, Y. Nagano, Y.-Q. Du, T. Ikeda and F. Toda, Tetrahedron Lett. 31 (1990) 5045. [13] K. Harata, Y. Nagano, H. Ikeda, T. Ikeda, A. Ueno and F, Toda, Chem. Commun. 2347 (1996). [14] H,J.C. Berendsen, J.P.M. Postma, W.F. van Gunsteren and J. Hermans, in B. Pullman (Ed.), Intermolecular Force, Reidel Dordrecht, The Netherlands, 1981. [15] DISCOVERUser Guide (version 2.9) Part I, Molecular Simulation Inc., 9685, Scranton Road, San Diego, CA. [16] C,A.G. Haasnoot, F.A.A.M. de leeuw and C. Altona, Tetrahedron 36 (1980) 2783.