Molecular Mechanics and Dynamics Studies on Amide-Modified

Chapter 8

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Molecular Mechanics and Dynamics Studies on Amide-Modified Backbones in Antisense Oligodeoxynucleotides R. M . Wolf, V. Fritsch, A. De Mesmaeker, J . Lebreton, and A. Waldner Central Research Laboratories, Ciba-Geigy Ltd., 4002 Basel, Switzerland

-

Replacing one or several phosphodiester linkages -O-PO -O-CH - in oligodeoxynucleotides by five structural isomers of an amide bond -NH-CO and two -CH - groups yields antisense oligonucleotides that retain the ability to form duplexes with complementary mRNA. Molecular mechanics and dynamics simulations reveal that the modified sequences can assume various conformations which allow for standard Watson-Crick base pairing with a complementary RNA strand without major strain or steric hindrance. The overall structural features and dynamical behavior of the modified R N A D N A hybrid duplexes are comparable although not identical to those of the wild-type RNA- DNA duplexes. 2

2

2

Antisense oligonucleotides represent a new class of potential therapeutical drugs. Their action is based on the repression of a defined protein by blocking specifically a portion of the corresponding mRNA (7-3). To be used as antisense agents, modified synthetic oligo(deoxy)nucleotides must retain the specificity towards complementary /wRNA (given by the base sequence) and should have similar or higher binding constants with /wRNA as compared to their wild-type analogues. Furthermore, they should be stable against degradation by nucleases and they should have an increased cell permeability with respect to natural oligonucleotides. Among the various possible modifications of natural oligonucleotides, backbone-modified nucleic acids look the most promising (4-6). Various approaches may be used to substitute the phosphodiester backbone which is exposed to the attack by nucleases. However, in order to allow for sequence-specific duplex formation with complementary RNA, the modified backbone must be able to adopt conformations which orient the bases in the best possible way for Watson-Crick base pairing. Although not necessarily required, this may be achieved most easily by keeping the number of bonds in the backbone as well as towards the nitrogen of the bases the same as in natural DNA, i.e., six bonds in the backbone and three bonds to the base nitrogen. Even for drastic changes in the backbone, like for example in 0097-6156/95/0589-0114$12.00A) © 1995 American Chemical Society Reynolds et al.; Computer-Aided Molecular Design ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


8. WOLF ET AL.

115

Amide-Modified Backbones

"peptide nucleic acids" (PNAs) in which the sugar portion is also substituted, the numbers of bonds are respected (7). The amide backbone modifications (see Figure 1) analyzed by molecular mechanics and dynamics in the scope of this work keep the sugar portions completely unaltered. Thus the modified backbone segment must be able to fold into a conformation which either resembles that of the natural phosphodiester or at least which leaves the orientation of the furanoses (and hence of the bases) essentially unchanged. The structures shown in Figure 1 were found to satisfy various criteria in order to be used in antisense compounds (8-14). Details about synthesis and experimental data are published elsewhere (9-14). Melting points T of duplexes with complementary R N A were obtained for different sequences with various numbers of substituted phosphate linkages (see Table I). m

Table I. Melting point differences between RNA* DNA duplexes with amidemodified DNA strands and the corresponding wild-type RNA- DNA hybrid duplexes. Average values over various sequences. modification

AT

amide 1

amide 2

amide 3

amide 4

amide 5

-2.8

-1.6

+0.4

0.0

-3.5

e r

m P modification (°C)

Amide modifications 3 and 4 have the highest T values (superior to the corresponding wild-type RNA- D N A hybrid duplexes in some cases). The other three amide modifications increasingly destabilize the duplexes in the order amide 2, 1, and 5. Although the experimental data clearly show differences between the various backbone modifications, these differences are not as spectacular as anticipated. Above all, an interesting similarity in the T values was observed between those amide modifications which can be regarded as isomers of the same trans double bond, namely amides 1 and 5, and amides 3 and 4, respectively. Without further proof, this may be taken as a first hint that the actual geometry of backbone modifications might be more relevant than the detailed electrostatics. The latter clearly change when reversing the orientation of the amide group, as is the case when passing from amide 1 to amide 5 or from amide 3 to amide 4. Also, it turns out that modifications directiy connected to one of the sugars (amide modifications 1, 2, and 5) have a negative influence on the stability of the duplexes formed with complementary RNA. In order to understand in more detail the structural features of the various amide backbone modifications, molecular mechanics (MM) and molecular dynamics (MD) studies were undertaken. m

m

Reynolds et al.; Computer-Aided Molecular Design ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

116

COMPUTER-AIDED MOLECULAR DESIGN

Computational Methods M M and M D simulations were carried out with the A M B E R all-atom force field (75) "total=

Σ

[l

4

-

Σ

+

bonds

4&

-

Σ - ^ H οο8( φ-φ )] torsions +


Μ

Ν

θ

οΐ

2

angles

ί

Ν

Μ2

0

(

+

\6

(1)

Σ Σ % i=l j>i Ν

Ν

ΣΣ

i j

\ J

332 δ, δ , +

ι=1 j>i

Σ Η-bonds /υ

J _ Dij 10

12

as incorporated in the software package Insightll 2.2.0/Discover 2.9 from B I O S Y M Technologies, San Diego, USA. Electrostatic energy contributions were evaluated by using partial charges and applying Coulomb's law. Partial charges were assigned by an acceptor-donor scheme which reproduces as closely as possible the original A M B E R charges (75) in the unmodified portions of the structures (unpublished work by Thacher, T., B I O S Y M Technologies, San Diego). The permittivity was adjusted by a distance-dependent dielectric function ε = 4 τ - , where is the distance separating two charges δ, and δ^. The use of a distance-dependent dielectric function was found appropriate to account for the absence of explicit solvent molecules and counterions (16-18). Specific 1-4 nonbonded interaction energy terms were reduced to 50% (79). No cut-offs were used. Conformational Analysis. For the conformational analysis, starting structures were generated from an initial Α-form RNA- DNA octamer duplex r(GA G)- d(CT C) (see Figure 2) by making the appropriate changes to introduce the desired backbone modification between the middle residues in the D N A strand. Note that "A-form" in this respect refers to the helical parameters, the backbone conformation, and the sugar puckering, i.e., all furanose units were initially in C3'-endo puckering mode. Obviously however, the sugars were free to adopt any energetically accessible puckering mode in subsequent M D simulations. Different conformers were generated by enforcing chosen backbone torsion angles incrementally by 30°, followed by a complete relaxation by conjugate gradient until the maximum derivative was < 0.1 kcal-mol^-Â' . The enforcement of a torsion angle φ to a predefined value ty was performed by applying an additional harmonic energy term E of the form 6

6

1

cstr

cstr

E

(2)

k

cstr = cstr (Φ "Φ^ίΡ A

where the force constant k was set to 1000 kcal-mol" -rad" . This procedure allows a scan through conformational space without an actual disruption of the duplex. cstr


WOLF ET AL.


pyΟ

Η—Ν

/

C3'-

/

\

CH


c=o ο

/ \

CH

C3'-

amide 1

amide 2

/ HC

C3'-

C3'0=C

2

Η—Ν

/

c=o

/

N-H

/ \

/ HC

o=c\

\

\

C=0 / C4'-

r

I

2

CH,

C4'-

/

Η—Ν

2

wild-type

C3'-

2

2

Ν—Η

\

C4'-

C4'-

CH, / C4'~

amide 3

amide 4

amide 5

CH

CH,

2

2

Figure 1. Amide-modified backbones replacing the wild-type phospho diester (top left) in antisense oligofdeoxynucleotides.

r 5

p

p

p

p

p

p

p

y

'7^ T T T T T T 7~

octamer for conformational analysis

G A A A A A A G C Τ Τ [Τ ; j l T T C , d 3 ' - ^ p ^ p ^ p t 2 ^ p - - p - " p " - " 5' J

r 5 L

p

p

p

p

J

p

L

p

p

p

p

p

p

p

p

3

7T T T T T T T T T T T T T 7~ '

G A A A A A A A A A A A A G C T T T T T T T T T T T T Ç ! , d3 ^p^p^*^p^*^p^*^p^*-^p -*- p- -p- -5' ,

J

L

i

i :

I4mer for molecular dynamics Figure 2. Structures used for conformational analysis (top) and molecular dynamics (bottom). * designates the replacement of a phosphodiester linkage by an amide modification.


COMPUTER-AIDED M O L E C U L A R DESIGN


118

Although the method cannot ensure the finding of all possible local minima, it yields a representative range of low-energy conformers to be considered further. Torsion angles around bonds directly connected to one of the sugar rings, i.e., either to C 3 ' in the case of residue i or to C 4 ' for residue M in Figure 3, were not enforced. The enforced torsion angles for the amide modifications were α and β for amides 1 and 5, ζ and α for amide 2, ζ and β for amides 3 and 4 (see Figure 3 and reference (20) for definitions). Note that the amide bond was always preset to either trans (180°) or cis (0°) and then let free to relax, i.e. deviating moderately from the planar structure. Although the cis amide is energetically less favorable in the isolated form, its occurrence was not excluded a priori in die modified duplex structures. Molecular Dynamics. The various local-energy-minimum conformers found during the conformational analysis were used as starting geometries for the M D runs, which were carried out on alternatingly modified R N A - D N A \4mer duplex structures r(GA G)-d(CT(T*T) TC), where * designates a specific amide-modified linkage in a defined starting conformation (Figure 2). The alternatingly modified structures were chosen for simulations because of the experimental feasibility of such backbones, for which modified dimers can be connected by standard nucleotide oligomerization techniques (27). Prior to molecular dynamics, the structures were completely minimized. M D simulations were then run in the NVT ensemble, keeping the temperature constant at 300 Κ by coupling to an external heat bath (22). One-femtosecond time steps were used for the numerical integration. The system was preconditioned by heating stepwise from 0 to 300 Κ over a period of 24 picoseconds: 2 ps at 50 Κ and 100 K , 4 ps at 150 Κ and 200 K , 5 ps at 250 K , 7 ps at 300 K . Trajectories were then recorded for 100 ps. Instantaneous coordinates were saved every 0.5 ps for subsequent analysis. 12

5

Results and Discussion Conformational Analysis. For each of the backbone modifications various local minima on the potential energy surface were found, sometimes differing only marginally in energy. Lowest-energy conformations for the different backbone-modified structures are shown in Figures 4 to 6. These figures represent the T*T sequence cut out of the octamer duplex without the base atoms. For amide 3 (Figure 5) and amide 4 (Figure 6) are depicted the three lowest-energy structures. Torsion angles for localenergy-minimum geometries are not listed explicitly here (see references (9-75) for more details). During subsequent M D computations these values are subject to either moderate oscillations or definite transitions to other domains. Note that all amide structures shown in Figures 4 to 6 have sugars with N-type puckering (between C 3 ' endo and 04'-endo), i.e., the torsion enforcement on the modified backbone linkages did normally not alter considerably the puckering mode from the C3'-endo starting geometry. A more detailed discussion of backbone torsion angle transitions and of the sugar puckering follows in the subsequent sections dealing with M D simulations. A comparison between the lowest-energy conformations of amide 1 and amide 5 (Figure 4), respectively of amide 3 and amide 4 (Figures 5 and 6) reveals a close geometrical resemblance between these structures that are the respective isomers of the same hypothetical trans double bond. The small differences can be attributed to the


8.


W O L F E T AL.


I C3'—C2'

i+1 .C3'-C2' Figure 3. Definition of torsion angles in backbone-modified oligonucleotides: X-Y- -Z$-W3-C4'- -C3'- -x£-Y-Z; 04'-Cl'-*>-Nl-C2 (according to reference (20). a

z

z

Figure 4. Lowest-energy geometries for the amide backbone modifications 2 (left), 1 (center), and 5 (right). The structures were cut out of the octamer duplex. Base atoms are not shown for clarity.



120


Figure 5. Three lowest-energy geometries for the amide 3 backbone modification: 3a (left), 3b +0.5 kcal-mot (center), 3c +2.9 kcal'mot (right). Energy values refer to the entire octamer duplex as shown in Figure 2. See also legends of Figure 4. 1

1

Figure 6. Three lowest-energy geometries for the amide 4 backbone modification: 4a (left), 4b +1.6 kcal'mot (center), 4c +3.8 kcal'mot (right). See also legends of Figures 4 and 5. 1


1

8. WOLF ET AL.

121


repulsion between the amide oxygen and the furanose ring oxygen (04') in amides 1 and 4, as compared to the corresponding attraction of the amide hydrogen to the 0 4 ' atom in amides 5 and 3, respectively. Overall, the similar lowest-energy backbone conformations corroborate the interpretation of the experimental results that geometrical considerations seem to govern the relative stability of the modified duplexes. In amides 1 and 5, the amide bond corresponds to the backbone torsion angle ζ which is thus forced to roughly trans. The other backbone torsion angles have to adjust to this "unnatural" conformation, resulting in an overall backbone arrangement not commonly found in nucleic acids, e.g., α and β in gauche plus (g ). In the amide 2 modification, β coincides with the amide bond. The lowest-energy conformer is found to adopt a backbone conformation similar to that in wild-type A - D N A , i.e, all backbone torsion angles automatically adjust to the standard confor mational ranges found in D N A once β is fixed to the "natural" trans range. In that sense, the amide 2 modification is the perfect geometrical match of a natural D N A backbone. In both amide 3 and amide 4, the amide bond corresponds to a. In standard D N A duplexes, α is generally in the gauche minus (g~) range and the torsion angle γ is in the gauche plus (g ) range. However, in another low-energy geometry, found also experi mentally in A - D N A crystals (25-25), both α and γ are in the trans range. The lowestenergy conformations induced by the modifications amide 3 and amide 4 are found to have [α,γ] in [f,r] with the other torsion angles adopting the same conformational ranges as in the alternative A - D N A structures mentioned above. Thus, both amide 3 and amide 4 modifications induce a backbone conformation found experimentally in X-ray studies on D N A and hence virtually free of strain.


+

+

Molecular Dynamics. It is experimentally established that in R N A - D N A hybrid duplexes, the R N A strand riboses stay in the C3'-endo puckering domain, the D N A strand deoxyriboses adopt an average puckering mode between 04'-endo and Cl'-exo, and the global helical parameters of the hybrid duplexes are closer to Α-form than to B-form (26-30). Considering furthermore the low-energy barriers for deoxyribose puckering transitions, the Α-form starting geometry seems appropriate for M D simulations. This was also verified in M D simulations of a wild-type RNA- D N A hybrid duplex r ( G A G ) - d(CT C) for which the experimental results were correctiy reproduced (57). The various modifications in different low-energy conformations were introduced alternatingly in the D N A strand of the \4mer R N A - D N A duplex (Figure 2). For the amide modifications, the resulting energy differences in the \4mer duplex between the distinct conformers were roughly proportional to the differences observed for a single modification in the octamer duplexes investigated in conforma tional analysis. This finding may be explained by a compensation of geometrical changes by the alternating wild-type phosphodiesters in such a way that consecutive amide modifications do not "feel" each other when separated by a natural linkage. M D results were analyzed with respect to backbone conformational transitions and to the behavior of the sugar puckering in the modified strands. Detailed helical parameters were not considered at this stage of the investigation. For a global overview, Figures 7 and 8 depict the \4mer duplex starting structures and the average dynamics geometries, with coordinates averaged over the 100 ps trajectories. These figures visualize qualitatively the fact that the amide modifications lead to stable duplexes in 12

12


122


which base pairing is more or less conserved on average, although momentary disruptions were noticed during the M D trajectories, an observation made also for the wild-type reference structure (structures on the left in Figures 7 and 8). In Table Π are listed time-averaged values (with standard deviations in parentheses) for the backbone torsion angles, the glycosidic torsion angle χ and the sugar puckering parameters Ρ and x (defined according to reference (32)) for the middle residues T*T of the amide-modified 14mer duplexes r(GA G)-d(CT(T*T) TC). Note that there was no substantial difference between the behavior of these middle dimers and the adjacent modification-linked dimers. A l l data reported in Table II were obtained starting from the lowest-energy conformations as shown in Figures 4 to 6. The corresponding data for an unmodified RNA- D N A duplex, obtained under identical conditions, are listed for comparison. m


12

5

Backbone Transition. D N A strands with amide modifications 1 and 3 oscillated around the lowest-minimum-energy conformation shown in Figures 4 to 6. This is evidenced by the small standard deviations for the backbone angles reported in Table II. For amide 3, starting from the second-lowest-energy conformer (3b in Figure 5), four out of five modified residues converted into the lowest-energy geometry (3a in Figure 5) during the 100 ps dynamics run. During this simulation period, no transition from 3a to 3b was observed. Since the conversion from 3b to 3a is readily observed, the involved barrier can be overcome on the 100 ps time scale. Also, the difference in potential energy between the two conformations is rather small (~ 0.5 kcal- mol" ). The absence of 3a -» 3b transitions has been attributed to the larger puckering amplitude observed in the 3a conformation, i.e., the overall entropy of the system increases when passing from 3b to 3a. Thus, the 3a conformation would be preferred over 3b for free energy reasons. A more detailed analysis on these transitions and their possible reasons will be given elsewhere (Wolf et al., submitted for publication). In amide 2 and amide 5 modified D N A strands, transitions in the modified backbone portions were observed even when starting from the lowest-energy geometry as depicted in Figure 4. The amide 2 modified strand underwent transitions [α,γ] from [g\g ] to [r,r] in various unmodified portions, but also in the amide-modified residues. This type of transition is commonly observed in molecular dynamics simulations on wild-type D N A - D N A and RNA- D N A duplexes (see e.g. references (57,3334). Its occurrence in the simulation of the amide 2 modified backbone portions underlines the geometrical similarity of the amide 2 modification and the natural phosphodiester linkage, already observed in the conformational analysis. In amide 5 modified strands α and ε in the modified part oscillated coherently between two conformational domains, the lowestenergy domain as depicted in Figure 4 and the next-lowest-energy domain found in the conformational analysis. Similarly, the amide 4 modified portions showed transitions between the low-energy structures 4a and 4b depicted in Figure 6. These transitions are related to changes in the torsion angles β and ζ, as seen also in the higher values for the M D standard deviation of these angles in Table II. 1

+

Sugar Puckering. The alternating character of the amide-modified backbones has an interesting effect on the overall sugar puckering scheme. Indeed, there are two different types of deoxyriboses, one having the modified backbone part attached to the C3' carbon (residue i in Figure 3) and one having the modified backbone sequence



8. WOLF ET AL.


Figure 7. Starting structures (top) and average dynamics geometries (bottom) of 14mer duplexes. From left to right: wild-type RNA - DNA, amide 1, 2, and 3a modified duplexes. The 5'-end of DNA or modified DNA strands is always at the left top of each duplex. The ribbons go through C3' as trace atom with CT being the plane atom.

Figure 8. Starting structures (top) and average dynamics geometries (bottom) of 14mer duplexes. From left to right: wild-type RNA'DNA, amide 4, and amide 5 modified duplexes. See also legend of Figure 7.


COMPUTER-AIDED MOLECULAR DESIGN

124

Table II. Time averages and standard deviations (in parentheses) of torsion angles and puckering parameters (in degrees) for the central dimer in the amide-modified DNA strand in Umer duplexes r(GA G)-d(CT(T*T) TC). 12

wild-type b


a>

β Y δ ε

ζ

Χ

c )

-70 ( l l ) -70 (12)

175 (10) 176 (10)

amide 1 -73 (12) d)

60 (14)

a)

5

amide 4a

a)

amide 2

amide 3a

amide 5

-71 (12)

-74 (11)

-78(11)

-75 (11)

-140 (64)

171 (8)

171 (9)

105 (40)

172 (9)

175 (10)

179 (8)

174 (9)

171 (9)

64 (7)

179 (10)

-149 (24)

-94 (21)

69 (9)

63 (9) 62 (11)

59 (9)

60(10)

58 (10)

59 (10)

61 (10)

62 (10)

127 (58)

173 (9)

178 (9)

67 (10)

108 (18) 108 (17)

98 (23) 90(14)

83 (14) 104 (27)

94 (19) 107 (25)

84 (16) 100 (23)

90(18)

-176 (8) -176 (9)

-178 (14)

-175 (8)

176 (9)

169 (9)

155 (44)

-175 (10)

-170 (10)

-166 (9)

-166 (10)

-172 (9)

-89 (12) -88 (11)

180 (11)

-71 (13)

-100 (18)

-137 (24)

-176 (10)

-83 (12)

-78 (14)

-79 (13)

-76 (13)

-79 (12)

-139 (16) -140 (15)

-134 (19) -139 (16)

-156 (12) -146 (15)

-156 (13) -154 (17)

-154 (11) -156 (15)

-145 (19) -139 (16)

110 (27) 112 (26)

82 (41) 93 (23)

66 (28) 88 (55)

86 (35) 103 (43)

61 (33) 90(40)

63 (40) 87 (31)

42(5) 42(6)

44(6) 46 (5)

41 (6) 40(6)

42 (6) 42 (6)

42(6) 42(6)

43 (6) 43 (6)

88 (18)

Using the lowest-energy geometry in Figures 4 to 6 as starting points. ' See Figure 3 and reference (20) for torsion angle definitions; the first line for each value refers to the residue with the amide-modified backbone portion attached at C3* (i in Figure 3), the second one to the residue with the modification attached to C4' (i+1 in Figure 3); ^ values in bold correspond to modified backbone portions; * Ρ is the phase angle of pseudorotation and x the maximum degree of puckering (see reference (32) for definitions). c )

d

e

m

attached to C4' (residue M in Figure 3). The corresponding puckering modes are found on the first and second line, respectively, in Table II. For all five amide modifications, these two types of deoxyriboses have a different average puckering behavior. In all cases the sugars with the modification attached at C3' (0 have smaller average values for Ρ than the sugars with the modified portion bonded to C4' (i+7). Considering that the difference of two degrees observed between the Ρ values of the middle residues in the wild-type RNA* D N A duplex reflects the random character of molecular dynamics, the corresponding differences in the amidemodified strands are significant (e.g. almost 30° for amide 4).



8. WOLF ET AL.

125


Furthermore, the sugar puckering scheme was found to depend also on the actual backbone conformation, and not only on the nature of the amide modification. Indeed, starting the dynamics trajectories from other low-energy conformers (not shown in Table II) leads to a different puckering scheme in some cases, as already reported in the previous section. The backbone conformation controls both the amplitude and the mean value of the phase angle of pseudorotation P. Furthermore, the puckering state of the sugar determines to some extent the torsion angle to the base χ. TTius the various substructures in the modified DNA strands are strongly interrelated and modifications to one part inevitably affect other portions. Details about this structural interdependence will be given elsewhere (Wolf et al., submitted for publication). The (unmodified) complementary RNA strands showed no unusual behavior in any of the amide-modified duplexes. All riboses remained confined to the C3'-endo puckering mode as has also been found for RNA strands during MD simulations of wild-type RNA- DNA hybrid duplexes (37). Conclusions The simulation results of alternatingly amide-modified DNA strands paired to complementary RNA sustain the concept that such modifications do not introduce considerable strain or steric hindrance. The amide-modified duplex structures behave quite similarly to the natural RNA- DNA hybrids (26-37) with allribosesin the RNA strand adopting the standard A-type puckering C3'-endo, whereas the deoxyriboses oscillate between the classical C3'-endo and C2'-endo puckering modes, with an average value concentrating around 04'-endo. Some modifications can adopt "natural" DNA conformations because the amide bond corresponds to a torsion angle which is trans (like β in the canonical ADNA, or α in the other low-energy conformation found in standard DNA (23-25)). Thus, amide 2, with the amide bond corresponding to β, adopts all torsion angles in the range of wild-type DNA. Furthermore, the transition [α,γ]from[g',g ] to [t,t], observed in MD simulations of wild-type DNA- DNA (33,34) or RNA- DNA (37) is also noticed for this modification. Both amide 3 and amide 4, with the amide bond corresponding to a, adopt the lowest-energy conformation with [α,γ] = [t,t]. Obviously, the simulations alone cannot explain the T differences observed between the five amides reported. These differences would have to be analyzed from the point of view of free energy, i.e., including entropy considerations. Such an approach is currently excluded considering the very large conformational space available to single strands. Still, the simulations have revealed structural details concerning possible backbone conformations and sugar puckering schemes which may be considered as useful hints for the design of further backbone modifications of this type. +

m

Literature Cited 1. 2. 3. 4. 5.

Uhlmann, E. and Peyman, A. Chem. Rev. 1990, 90, 543. Crooke, S.T. Annu. Rev. Pharmacol. Toxycol 1992, 32, 329. Cook, P.D. Anti-Cancer Drug Design 1991, 6, 585. Quaedflieg, P.J.L.M.; van der Marel, G.A.; Kuyl-Yeheskiely, E.; van Boom, J.H. Recl. Trav. Chim. Pays-Bas 1991, 110, 435. Vasseur, J.-J.; Debart, F.; Sanghvi, Y.S.; Cook, P.D. J. Am. Chem. Soc. 1992, 114, 4006. Reynolds et al.; Computer-Aided Molecular Design ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

126 6. 7. 8. 9.


10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

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Molecular Mechanics and Dynamics Studies on Amide-Modified

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