Voiume3 no.8 Augusti976
Nucleic Acids Research
Structural studies on the two forms of 8-bromo-2',3f-O-isopropylideneadenosine
Satoshi Fujii, Takaji Fujiwara and Ken-ichi Tomita
Faculty of Pharmaceutical Sciences, Osaka University, 133-1 Yamada-kami,
Suita, Osaka 565,Japan
Received 25 May 1976
ABSTRACT
The crystal and molecular structures of two forms of 8bromo-2',3'-O-isopropylideneadenosine have been determined by
X-ray methods. In one form, the molecular structure has planar
conformation in the sugar moiety and no intramolecular hydrogen
bond. On the other hand, the molecular structure of the second
form has C(2')-endo conformation and an intramolecular hydrogen
bond. No stacking interaction between adjacent bases is found in
either form, but two modes of the base-pairing hydrogen bond
exist in the second form.
INTRODUCTION
Halogenated purines and pyrimidines have been widely studied
because of therapeutical or general biochemical interest. Ikehara
et al.
have reported on the effect of halogenation of purine
ribonucleoside diphosphates on their reaction with polynucleotide
phosphorylase and described that such halogenated polynucleotides
might have a somewhat different but less stable conformation than
usual polynucleotides by virtue of its physical and biochemical
specificity.
This might be due to the conformational change
around the glycosidic bond, though the hydrogen bonding sites of
bases remain unchanged.
By a novel method
for the 8,5'-cyclization of halogenated
purine nucleosides, 8,5'-anhydro-8-oxyadenosine
has been synthe-
sized from 8-bromo-2',3'-O-isopropylideneadenosine
material.
as starting
However, such a cyclization reaction does not occur
when 8-bromoadenosine is used, of which the crystal and molecular
structure has been elucidated by Sobell et al. .
It seems,
therefore, reasonable that this cyclization specificity may be
attributed to the conformational difference of the sugar moiety.
In this paper, the crystal and molecular structures of two
forms of 8-bromo-2',3'-O-isopropylideneadenosine
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
obtained by the
1985
Nucleic Acids Research
same crystallization procedure are described and also stereochemical considerations are given to correlate conformational change
and reactivity of halogenated polynucleotides.
MATERIAL AND METHOD
The compound, 8-bromo-2',3'-0-isopropylideneadenosine, was
kindly supplied by Dr. M.Ikehara.
It was dissolved in n-butanol-
saturated aqueous solution at 50°C.
The distinct crystalline
forms (crystal I and crystal II) weTe separately obtained by slow
evaporation at room temperature.
The former is obtained by
relatively fast evaporation as colourless prisms and the latter
by a slower evaporation from dilute solution as colourless plateshaped crystals.
X-ray analysis : The unit cell and space group of each form were
determined by 30° precession photographs.
More accurate cell
constants were remeasured by a diffractometer.
Both densities
were measured by the flotation method with a mixture of carbon
tetrachloride and ethylene bromide.
The final crystallographic
data are shown in Table I, and the atomic numbering is shown in
Fig. 1.
Table I. Crystal data of 8-bromo-2',3'-0-isopropylideneadenosine
I
Crystal
Chemical Formula
a (A)
b (A)
c (A)
6 C)
Space Group
Z
Dm Cg/cm')
Dx (g/cm 1 )
Hoi. wt.
11
Ci,l,,S, O.Br
10.06 : 0.01
18.55 ± 0.02
8.20 : 0.01
C I H, 1 N, O.Br:H2O
19.526 : 0.012
9.212 s 0.007
9.012 : 0.005
99.37
P2i2i2i
C2
4
1.67 ! 0.01
1.68
386.2
4
1.68 i 0.01
1.68
404.2
Fig.l. Atomic numbering used in this vork.
1986
Nucleic Acids Research
Intensity data for each form were collected on a full
automatic four-circle diffractometer (Rigaku Denki Ltd.)-
1094
independent reflections for crystal I with dimensions of about
0.10x0.30x0.40 mm 3 were measured with the w-28 scan technique
only in the range of £ = 0 ^ 4 because of gradual damage by X-ray
radiation, using Ni-filtered Cu-Ka radiation.
(dimension3; 0.05x0.30x0.30 m m 3 ) ,
For crystal II
1200 reflections were measured
in the range of 0 < sin6/X < 0.55 by a similar method.
absorption correction was applied.
No
The absolute scale and
overall temperature factors were determined by the usual
Wilson's statistics.
Both structures were solved by the heavy-atom method.
Three-dimensional sharpened Patterson functions were calculated
and Br-Br vectors between adjacent molecules related by symmetry
were easily found.
The successive Fourier synthesis revealed the
locations for all non-hydrogen atoms.
With anisotropic
temperature parameters to all non-hydrogen atoms, the structures
were refined by block-diagonal least-squares method.
At this
stage, the discrepancy factor, R=I || Fo |-| Fc \\/l | Fo | , dropped to
0.130 for crystal I and 0.067 for crystal II.
In the case of
crystal II, a difference Fourier synthesis was then carried out,
which gave the positive peaks at the proper positions for all
hydrogen atoms except six of the isopropylidene group.
After
several cycles of refinement including the hydrogen atoms, the
discrepancy factor for crystal II was reduced to 0.061.
NMR study ; The NMR spectra of the compounds were obtained with
100 MHz high resolution NMR spectrometer (Hitachi).
The sample
was dissolved in d 6 -DMSO and the final concentration was 40mg
in 4ml.
RESULTS AND DISCUSSION
Final co-ordinates for the two forms of 8-bromo-2',3'-0isopropyiideneadenosine are shown in Table II (crystal I) and in
Table III (crystal II).
The bond lengths and angles of the two
forms are shown in Fig. 2 (crystal I) and in Fig. 3 (crystal II).
Owing to the low accuracy of the final structural parameters,
not much discussion on the bond lengths or angles is given.
Molecular structures of the two forms projected on the respective
purine plane are shown in Fig. 4 (crystal I) and in Fig. 5
1987
Nucleic Acids Research
Table I I . F i n a l c o - o r d i n a t e s of c r y s t a l I of 8-bromo-2J3'-O-isopropylideneadenosine and standard deviations in parentheses(xlO )
x/a
Atom
2/C
y/b
0.0933(02)
0.2900(05)
Br
-0.0108(03)
N(l)
C(2)
N(3)
C(4)
C(5)
C(6)
N(6)
N(7)
C(8)
N(9)
C(l')
0(1')
C(2')
0(2')
C(3')
0(3')
C(4')
C(5r)
0(5')
C(I)
C(II)
C(III)
0.2523(22)
0.2472(38)
0.1910(22)
0.1518(22)
0.1530(19)
0.2067(21)
0.2213(31)
0.0967(17)
0.0674(29)
0.0935(23)
0.0758(32)
-0.0273(19)
0.2042(29)
0.2512(22)
0.1538(30)
0 . 1 8 0 7 (19)
- 0 . 0 0 3 4 (29)
-0.0263(26)
-0.1692(18)
0.3051(43)
0.0658(30)
0.2102(31)
0.1526(10)
0.2223(18)
0.2448(13)
0.1896(12)
0.1171(10)
0.0966(15)
0.0263(13)
0.0755(09)
0.1221(13)
0.1896(11)
0.2562(14)
0.2950(09)
0.3027(18)
0.3203(10)
0.3766(14)
0.4274(09)
0.3661(13)
0.3778(19)
0.3831(09)
0.4289(17)
0.3834(16)
0.3901(13)
0.9668(35)
0.9229(58)
0.7697(40)
0 . 6 7 4 7 (43)
0.7175(43)
0.8742(40)
0.9224(36)
0.5959(33)
0.4873(49)
0.5302(35)
0.4190(43)
0.4704(29)
0.4220(48)
0.2670(33)
0.4931(48)
0.3644(30)
0.5223(38)
0.7045(44)
0.7183(30)
0.1209(55)
0.1153(50)
0.2122(47)
T a b l e I I I . F i n a l c o - o r d i n a t e s of c r y s t a l I I of 8-bromo-2',3 ' - 0 Atom
Br
N(l)
C(2)
N(3)
C(4)
C(5)
C(6)
N(6)
N(7)
C(8)
N(9)
C(l')
O(l')
C(2')
0(2')
C(3')
0(3')
C(4')
C(5')
0(5')
C(I)
C(II)
C(III)
O(H2O)
1988
x/a
0.1764(01)
0.0875(05)
0.1572(07)
0.2048(06)
0.1772(06)
0.1065(06)
0 . 0 6 0 7 (07)
-0.0083(06)
0.0949(05)
0.1576(07)
0.2094(05)
0.2843(06)
0.3016(05)
0.3310(06)
0.3544(05)
0.3962(07)
0.4354(05)
0.3720(06)
0.3671(07)
0.3369(05)
0.4215(07)
0.4163(09)
0.4749(08)
0.2920(08)
y/b
z/c
0.1000(04)
0.3108(14)
0.3102(20)
0.2600(17)
0.2335(16)
1.2746(01)
0.6096(12)
0.6165(16)
0.7345(12)
0.8537(13)
0.2415(17)
0.2849(16)
0.2931(16)
0.2016(15)
0.1618(19)
0.8663(14)
0.7333(15)
0.1797(14)
0.1490(16)
0.0446(11)
0,2871(18)
0.3464(13)
0.2198(22)
0.1760(14)
0.0740(16)
0.0788(25)
0.2098(15)
0.2778(22)
0.1901(26)
0.4090(24)
0.4318(18)
0.7266(14)
1.0099(12)
1.0797(16)
0.9905(11)
1.0326(15)
0.9299(11)
1.0268(15)
1.1692(11)
0.9764(17)
0.1227(12)
0.8995(15)
0.7287(16)
0.6680(11)
1.2317(15)
1.3709(20)
1.2559(21)
0.4414(15)
Nucleic Acids Research
o
Fig.2. Bond lengths(in A) and bond angles
(in degrees) of crystal I of 8-bromo-2\3'•
O-isopropylideneadenosine. The average
standard deviations are 0.05A for bond
length and 3° for angle.
Fig.3. Bond lengths(in A) and bond angles
(in degrees) of crystal II of 8-bromo-2J3'
O-isopropylideneadenosine. The average
standard deviations are 0.021A for bond
length and 1° for angle.
1989
Nucleic Acids Research
Fig.5. Molecular structure of
crystal II. The dotted line
indicates the intramolecular
hydrogen bond.
Fig.4. Molecular structure of
crystal I.
(crystal II).
The torsion angle x around the glycosidic bond (this angle is
O(l')-C(l')-N(9)-C(8). 4 ) is 252° for crystal I, while it is 239°
for crystal II and similar to that found in 8-bromoadenosine (x=
240°).
Table IV.
In Table IV, the torsion angles x °f 8-bromoadenosine
Molecular conformation of 8-bromoadenosine
compound
torsion
angle
8-bromoadenosine
dihedral
angle between
base 6 sugaT
240°
77°
sugar
hydrogen bond
C(5')-O(5')
conformation
N(3)~H-O(5')
orientation
C(2')-endo
(0.56A)
2 74A
gauche-gauche
- —
gauche-trans
8 -Br-2!J'-IPD-adenosine a
(this work) crystal I
252°
78°
planar
8-Br-2 J 3'-IPD-adenosine
(this work) crystal II
239°
77°
C(2')-endo
(0.40A)
2 78A
gauche-gauche
244°
74
C(2')-endo
(0.65A)
2 98A
gauche-gauche
8-Br-2'-O-TPS-adenosine^
crystal I
Mol. 2
236°
78°
C(2')-endo
(0.59A)
3 04A
gauche-gauche
8-Br-2'-0-TPS-adenosine 6
crystal II
250°
76°
C(2')-endo
(0.54A)
2 85A
gauche-gauche
8-Br-3•-O-TPS-adenosine 5
240°
78°
C(2')-endo
(0.59A)
2 89A
gauche-gauche
8-Br-2'-0-TPS-adenosine b S
crystal I
Mol. 1
(a)
IPD»
O-isopropylidene
(b)
TPS»
triisopropylbenzensulfonyl
(c)
the orientation of the C(5')-O(5') bond with respect to the ring bonds C(4')-O(l')
and C(4')-C(3')
1990
Nucleic Acids Research
derivatives are listed together with other conformational
characters.
From these values, the 8-bromoadenosine derivatives
reported thus far all have the syn conformation as well as
8-bromoguanosine
which may be due to the steric repulsion
between the 8-substituted bulky Br atom and the sugar moiety.
It
is a significant difference between crystal I and crystal II that
o
the N ( 3 ) — H-0(5') intramolecular hydrogen bond (2.78A) exists in
crystal II but not in crystal I.
This difference may cause the
difference in sugar conformation between crystal I and crystal II.
The ribose ring of crystal I is nearly planar, while that
of crystal II is in the C(2')-endo conformation where C(2') is
o
displaced 0.40A out of the best plane through the remained four
atoms of the ring, similar to that found in 8-bromoadenosine
O
(C(2') deviating by 0.56A).
In crystal I, the orientation of the
C(5')-O(5') bond with respect to the bonds C(4')-0(l') and
C(4')-C(3') is gauche and trans with angles of 78° and -169°,
respectively.
The gauche-trans conformation is also seen in
0
cyclic cytidine-2',3'-phosphate
conformation of the ribose ring.
, which has also a planar
On the other hand, the orienta-
T
tion of the C(5')-O(5 ) bond in crystal II is gauche-gauche with
angles of -76 C and 42°, respectively, which is very common in
nucleosides with syn conformation and having an intramolecular
hydrogen bond.
The orientation of the C(5')-O(5') bond has a
strong influence upon the N(3)«~H-0(5') intramolecular hydrogen
bond formation.
The isopropylidene ring formed by 0(2'), C(2')
C(I), C(3') and 0(3') also affects the sugar conformation but
this extra ring does not exist in natural nucleosides.
However,
there is in nature such a compound, 2',3'-cyclic AMP with a
similar five-membered ring connecting C(2') and C(3'), and the
detailed conformational study of the isopropylidene ring may be
useful for investigation of structure-function relationships in
biologically important cyclic mononucleotides.
In crystal I,
atoms, 0(2'), C(2'), C(3'), and 0(3') are coplanar and C(I) is
o
puckered by 0.34 A toward the sugar ring, similar to that found
in cyclic cytidine 2',3'-phosphate, uridine 2',3'-0,0-cycloo
phosphorothioate and 2 ',3' -0-isopropylidene-3,5'-cycloadenosine
. The distances between the two methyl carbon atoms
o
o
attached to C(I) and 0(1') are 3.46 A and 5.06 A.
The former 1991
Nucleic Acids Research
value is very close to the sum of the van der Waals radii of
o
an oxygen atom and a methyl group, 3.4 A.
In crystal II, the
o
distances of the two methyl groups to 0(1') are 4.44 A and
5.31 A.
The crystal structure of crystal I projected along the caxis is shown in Fig. 6 where the broken lines indicate the
Fig.6- The molecular packing of crystal I
of 8-bromo-2;3'-O-isopropylideneadenosine
viewed down along the c-axis. The dotted
lines indicate the hydrogen bonds (A).
hydrogen bonds.
Adjacent molecules are connected to each other
by N(6)-H"*N(7) hydrogen bonds (2.99 A) to form ribbons running
parallel to the c-axis and related by two-fold screw axes, and
the base planes lie almost parallel to the b-axis.
The mode of
packing and hydrogen bonding of the molecules in crystal II
viewed down the b-axis is shown in Fig. 7 which indicates the
appearance of two types of direct hydrogen bonded base-pairing.
One is the N(6)-H—N(l) hydrogen bond (3.17 A) which is also
seen in the complex of 9-methyladenine with l-methyl-5-bromouracil 1 1
(designated as B type).
The other one is the
N(6)-H«"N(7) hydrogen bond (3.24 A) which is also observed in
adenine hydrochloride 12 and poly A (acidic form) 1
A type).
1992
(called as
Nucleic Acids Research
Fig.7. The molecular packing of crystal II
of 8-bromo-2', 3'-0-isopropylideneadenosine
viewed down along the b-axis. The dotted
lines indicate the hydrogen bonds (A).
Table V shows the base pairing of 8-bromoadenine
derivatives thus far determined by X-ray methods.
The B type
pairing is also found in the case of 8-bromo-2'-0-triisopropylbenzenesulfonyladenosine
, while the A type found in
crystal II is seen in some complementary base-paired complexes
containing 8-bromo-9-ethyladenine.
It is of particular interest
that two types of hydrogen-bonded base pairing exist in a crystal.
The dihedral angle between two hydrogen-bonded base planes is 37°
in both types indicating a propeller-like twist between hydrogenbonded bases.
This sort of angular deviation results in an
elongation of the hydrogen bond lengths.
No tendency of base
stacking parallel to each other exists in either crystal.
The two distinct sugar conformations found in this compound,
planar and C(2')-endo, are also observed in 2',31-O-isopropylidene-3,5'-cycloadenosine
(planar sugar conformation) and in
2-methyl-8-bromo-2',3'-O-isopropylideneinosine
puckering
(C(2')-endo sugar
). Therefore, these might be regarded as the preferred
1993
Nucleic Acids Research
Table . .
The pairing between 8-bromoadenine residues (hydrogen bond distances and angles)
compound
space
group
pairing type a
donor acceptor
distance
(A)
angle
C)
anglec
P2,/c
8-Br-9-Et-adenine" (in complex
with l-Me-5-Br-uracil)
8-Br-9-Et-adenine " (in complex
pi
with 8-Br-9-Et-hypoxanthine)
8-Br-9-Et-adenine"
Mol. 1 Pi
(in complex with phenobarbital)
8-Br-9-Et-adenine
Mol. 2 Pi
(in complex with phenobarbital)
A(i) N(6)-H-N(7)
3. 03
132
0
B(i) N(6)-H-N(l)
3. 01
119
0
A(i) N(6)-H-N(7)
3. 09
136
0
A(i) N(6)-H —N(7)
3 . 02
138
0
8-Br-2',3'-IPD-adenosine
crystal II
( t h i s work)
g-Br-2 1 -0-TPS-adenosine s
crystal I
Mol. 1
8-Br-V -O-TPS-adenosine s
A(2) N(6)-H-N(7)
B(2) N(6)-H-N(l)
B(2) N(6)-H-N(l)
3. 24
3. 17
126
112
121
37
37
24
C(2,)N(6)-H-N(1)
N(6)-H —N(7)
3. 06
3. 20
(a)
(b)
(c)
C2
P2,2,2
P2 1 2,2,
3 . 09
129
116
TYPEC
TYPEB
A,B,C see right
(1) ; inversion monad
(2) ; rotation diad
screw diad
(2 0 ;
34
C(6)-N(6)-N(7 or 1)
dihedral angle between
pairing bases
conformations even in ribose rings having an isopropylidene group
attached.
It is assumed that the potential energy barrier
between two sugar conformations is rather low, and the lattice
energy and/or intramolecular hydrogen bond energy are sufficiently large to overcome the barrier and to allow the interconversion.
The coupling constant (J-ji.i') °^ 8-bromo-2 ' , 3 '-0-iso
propylideneadenosine in the NMR spectra is 2.75cps and that of
8-bromoadenosine
is 6.50cps.
The approximate dihedral angles
and calculated coupling constants for some puckered
ring conformations
are summarized in Table VI.
5-membered
It may be seen
that the observed values of coupling constants suggest a C(2')endo sugar conformation for 8-bromoadenosine and a planar one
Table VI. Theoretical dihedral angles and coupling constants
for some puckered 5-membered ring conformation
Ring conformation
C(2'D-endo
C(2')-exo
C(3')-endo
C(3')-exo
planar
1994
H(l' )-H(2')
Angle Calculated
n
155
83
97
140
120
coupling
constant (cps)
8.3
0
0
5.8
2.1
Nucleic Acids Research
for 8-bromo-2',3'-0-isopropylideneadenosine.
Although it is
conceivable that the actual sugar ring conformation in solution
is the time averaged mixture of a puckered and a planar one, the
NMR results strongly support that the planar sugar conformation
of 8-bromo-2',3'-0-isopropylideneadenosine
found in crystal I
is preserved in solution.
Purine nucleosides or nucleotides have to have some
restraints on the torsion angle \, around the glycosidic bond.
There are three distinct allowable regions for the torsion
angle x, 3 ^ 5 5 ° , 120^123° and 2 1 0 ^ 2 5 8 ° , provided X-ray results
obtained are taken into account.
In the case of 8-bromoadenosine
derivatives, the bulky substituent at the 8-position of the
purine base may prevent the anti-conformation of x = 3 ^ 5 5 ° .
fact, the molecular conformations of 8-bromoadenosine
In
derivatives
have the syn conformation in the third region of x (210^258°) as
shown in Table IV.
(211°), formycin hydrobromide 20 (212°) , 6-thio-
deoxyguanosine
purine riboside
A similar conformation was also found in
21
(217°, 225°), 3'-O-acetyladenosine22
and 3',5'-cyclic adenosine monophosphate
(227°)
( about 258°).
The
nucleosides having the torsion angle x characteristic of the syn
region are remarkable because the sugar conformation is generally
C(2')-endo and the intramolecular hydrogen bond is formed between
N(3) and 0(5') except in crystal I with a planar conformation and
3',S'-cyclic AMP with a C(4')-exo puckered conformation.
On the
other hand, the bulky substituent at the 8-position of the purine
base has no effect on the base-pairing in adenosine derivatives
and as shown in Table V, three types of base-pairing were found
in the crystals.
The decrease of the stability in polynucleo-
tides containing such analogues probably arises from the
distinction of the torsion angle x but not from the alteration of
hydrogen bonding.
The difference in cyclization reaction between 8-bromoadenosine and 8-bromo-2',3'-0-isopropylideneadenosine
cited in
the introduction might be explained by comparing the different
sugar conformations in each compound.
By rotation around the
glycosidic bond, provided that the sugar conformation is fixed,
the shortest interatomic distance between C(8) atom of purine
o
and C ( 5 ' ) of r i b o s e i s 4.25A in t h e former compound
(C(2')-endo)
1995
Nucleic Acids Research
and 3.66A in the latter (planar).
Therefore, atom 0(5') of the
ribose in the former is too far away to attack the 8-position
of the purine by an S.,, reaction.
On the other hand, atom 0(5')
in the latter is able to attack the position to allow the
cyclization reaction by virtue of the short distance between
C(S') and C(8).
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