volume 2 number 8 August 1975
Nucleic Acids Research
Polynucleotides. XXXI1. Synthesis of AUG analogs containing 8,2'-Scycloadenoslne, 8,5'-S-cycloadenosine, 8-bromoadenosine, 8-oxyadenosine
and formycin in the first position of the codon
Morio Ikehara, Takeo Nagura and Eiko Ohtsuka
Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka,
Japan 565
Received 18 June 1975
ABSTRACT
Five AUG analogs having 8,2'-S-cycloadenosine (I), 8,5'-S-cycloadenosine (II),
8-bromoadenosine (III), 8-oxyadenosine (IV) and formycin (V) in the first position of
ApUpG were synthesized. 3'-Phosphates of I, II and V were synthesized by phosphorylation using cyanoethylphosphate and DCC. In the case of II, 2',3'-cyclic
phosphate was directly obtained. 3'-Phosphates, thus obtained, were properly protected on the 2'-OH and/or the N6-amlno group and condensed with U(OBz)pGiBu(iBu)2
using DCC to give ApUpG analogs. Some properties on paper chromatography and
electrophoresis, and the UV and CD spectra of these trinucleoside diphosphates are
reported.
We have previously synthesized 2 ' 3 analogs of the trinucleoside diphosphate
ApUpG, which is known^ to be the codon for methlonine and formylmethionine in the
protein biosynthesis system.
These analogs had 8,2'-O-cycloadenoslne or 8,5'-O-
cycloadenosine in place of the adenosine residue of the codon and activities in
Nirenberg's system^ for stimulating the binding of methionyl-tRNA to ribosomes
were observed.
In order to investigate the relationship of the structure of the first nucleotide
in the codon with its function we have now synthesized five AUG analogs in which A
had various base torsion angles (<£cn)6.
We chose 8,2'-S 7 and 8,5'-S-cycloadeno-
0
sine , because the exact torsion angles of these nucleosides have been elucidated
by X-ray crystallography^.
We also wanted to incorporate 8-bromo- 1 ^ and
8-oxyadenosine11 into the trinucleotide, where the base is confirmed to be in the syn
conformation as shown by X-ray 1 2 studies and CD spectra 1 3 .
cin was known to have a flexible torsion angle being in the syn
ary
15
The antibiotic formy14
or syn-anti bound-
region according to the nature of the medium.
When 8,2'-S-cycloadenosine (As) (I) was protected at the 5'-OH with the mono-
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Nucleic Acids Research
methoxytrityl (MMTr) group and phosphorylated with cyanoethylphosphate and DCC 16 ,
the phosphorylation occurred preferentially at the 6-NH2 group.
Therefore, the com-
pound (I) was fully acetylated and then treated with N NaOH In pyrldlne-water at 0° to
give N 6 -acetylA s (VI).
Compound VI was converted to the MMTr derivative (VII) and
reacted with cyanoethylphosphate and DCC.
Upon removal of the acetyl group MMTr-
AS 3'-phosphate (VIII) was obtained In a yield of 69%.
A s p(IX) was totally resistant to
snake venom 5'-nucleotldase hydrolysis In conditions, which gave hydrolysis of As 5'phosphate 17 .
The structure of VIII was confirmed and some of its physical properties
were examined.
NHAc
NHAc
>
(
(XII)
1346
X)
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Br
Br
Ac2O
Aci
( I l l ) R= H
0
(XIV) R=-P-OH
iAc
(XIII)
Bz.,0
6
(xix)
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Nucleic Acids Research
8,5'-S-Cycloadenoslne 3'-phosphate (sAp) (X) was obtained by phosphorylation
with cyanoethyl phosphate and DCC.
However, in this case treatment of the phos-
phorylatlon product with dilute ammonia gave the 2',3f-cycllc phosphate of 8,5'-Scycloadenosine (XI) only, which was subjected to hydrolysis with RNase M 1 8 to give
the 3'-phosphate (sAp) (X) in a yield of 51%.
These 3'-phosphates (VIH and X) were
treated either with acetic anhydride or with benzoic anhydride in the presence of triethylammonlum salts and the protected nucleotides were used for the condensation
reaction.
8-Bromoadeflosine 3'-phosphate (BrAp) (XIII) was obtained by the pro-
cedure described by Ikehara and Uesugi 10 in a yield of 45% from adenosine 2'(or 3f)
monophosphate.
When BrAp was treated with ace^c anhydride or benzoic anhydride* 9
to obtain the protected compound (XIV), S-oxy-N^'.S'-triacyladenoslne 3'-phosphate (XV) was also obtained according to the reaction conditions.
In the synthesis of formycin 3'-phosphate (Fp) (XVI), formycin 20 (V) was first
monomethoxytritylated and then treated with cyanoethylphosphate and DCC in pyridine-DMF solution.
I^-Dimethylaminomethylene-MMTr-formycin 2',3 f -cyclic
phosphate (XVII) was obtained in a yield of 92%. The cyclic phosphate XVII was
then treated with acetic acid and ammonia and then recyclized with DCC.
ted Fp> (XVIII), thus obtained was hydrolyzed with RNase T 2
21
Unprotec-
to give Fp (XVI).
The compound XVI was protected on N&, 2* and 5' by treatment with benzoic anhydride
to give compound XIX.
The properly protected 3'-phosphate was condensed with
2'-O-benzoyluridylyl(3'-5 t )-N 2 , a'.S'-triisobutyrylguanosine (U(OBz)p-GiBu(iBu)2, XX)
using DCC as the condensing reagent.
After the reaction, protecting groups were
removed by treatment with 80% acetic acid and methanolic ammonia.
Purification of
the product on DEAE-cellulose and Sephadex columns gave ASpUpG (XXI), sApUpG
(XXII), BrApUpG (XXIII), HOApUpG (XXIV) and FpUpG (XXV) respectively.
Struc-
tures of these trinucleoside diphosphates were elucidated by enzymatic digestion
using snake venom phosphodiesterase and RNase to give correct ratios of component
nucleosides and nucleotides as shown in the Experimental section.
Ultraviolet absorption spectra of these trinucleotides are shown in Figs 1-5 and
Table 1.
All trinucleotides have per residue values of 8,000-11,000, which seems to
be reasonable for this type of trinucleotide.
Hyper- and hypochromicity calculated
from values of each trinucleotide compaxed with those obtained after phosphodiesterase digestion are listed in Table II. Although these values are variable for different
nucleotides and wavelengths, the values at Xmax fall into a range of 7-3 to 15-7
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Nucleic Acids Research
i) DCC
Protected
ApUpG
(XXI)
ApUpG
(XXII)
BrApUpG
(XXHE)
HOApUpG
(XXIV)
S
ii) Deprotect,
Nucleoside
3-Phosphates
FpUpG
iBuO OiBu
comparable with that of AUG. (8>
(XXV)
(XX)
Discussions on these physical properties
together with CD spectra will be reported in another paper 22 .
EXPERIMENTAL23
5'-Monomethoxytrityl-8,2'-S-cycloadenosine
8,2'-S-Cycloadenosine7 (562 mg, 2 mmoles) and MMTrCl (740 mg, 2-4
mmoles) were dissolved in DMF (50 ml).
The solution was heated at 80-90°C for
4 hr and kept at room temperature overnight.
tion of the reaction showed a spot Rf 0*45.
TLC(CHCl3-EtOH, 10:1) examina-
The mixture was poured in ice-
Fig. 1
exlO"4
1.0
0.5
220
240
260
280
300
Wavelength (nm)
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Fig. 2
220
240
260
280
300
Wavelength (run)
Fig. 3
-4
1.0 •
0.5
220
240
260
Wavelength
1350
280
(mi)
300
Nucleic Acids Research
Fig. 4
-4
1.0
0.5
220
240
260
280
300
320
300
320
Wavelength (nm)
0.2
220
240
260
280
Wavelength (nm)
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Nucleic Acids Research
water containing 2% NH3.
H2O.
Precipitates were collected by filtration and washed with
The residue was dissolved In pyrldine (2 ml), evaporated twice with added
pyrldine, and precipitated as an amorphous powder by pouring into n-pentane.
8,2'-S-Cycloadenosine (1*0 g, 4-27 mmoles) was dissolved in pyridine
(50 ml) by slight heating.
To the solution acetic anhydride (2-6 g, 25-6 mmoles)
was added and It was heated at 90° for 2 hr.
The mixture was evaporated In vacuo,
pyridine and acetic anhydride were added again, and heated at 100° for 2 hr.
The
mixture was evaporated to a residue, which was taken up in a small amount of pyridine.
The pyridine solution was poured Into Ice-water and extracted with CHCI3.
The CHCI3 layer was washed with NaHCC>3 aq and H2O, dried over MgSO4, and
evaporated.
The oily residue was dissolved In a small amount of pyrldine and added
to n-pentane to cause precipitation of an amorphous powder.
Yield was 1 -2 g (75%).
Anal.Calcd. for C 18 H 19 O 7 S H2O: C, 46-25; H, 4-53; N, 14-98.
H, 4-39; N, 14-45.
Found: C, 46-43
NMR: (CDCI3, ppm) 1-92 (s, 3H, 3'-OAc), 2.-18 (s, 3H, S'-OAc),
2-47 (s,6H,N-Ac2), 4-22 (tri, 2H, 5'-H), 4-49-4»69 (m, 1H,4'-H), 4-41 (d, 1H.21H,Ji'-2'=5-4 Hz, j2'-3 f =2 Hz), 5-28 (t, 1H, 3'-H, 31-H,j2t-3'=2 Hz, j3'-4'= 5 Hz),
6-67 (d, 1H, l t -H,J 1 .. 2 <=5-4 Hz), 8-83 (s.lH, 2'-H).
N 6 ,3',5'-Q-Triacetyl-8,2'-S-cycloadenostne
Tetraacetyl compound (1 -2 g), obtained as above, was dissolved in 50% pyridine
(20 ml) and cooled at 0°.
Ice cooled N NaOH (10 ml, ca 2 equiv) was added.
5 min at 0°, the mixture was neutralized with IN HCl and evaporated in vacuo.
After
The
residue was dissolved In pyridine, insoluble material was removed by filtration and
the filtrate was evaporated.
into n-pentane.
The residue was dissolved in ethyl acetate and poured
Amorphous powder obtained in a yield of 1 -0 g (91%).
NMR: 1-94
(s, 3H, 3'-OAc), 2-18 (s, 3H,5'-OAc), 2-56 (s, 3H, NHAc), 4-19 (s, di, 2H,C5>-H2),
4-51 (m,lH,C 4 -H), 4-92 (dd, 1H,C2.-H), 5-32 (t, lH.Cg-H), 6-64 (d, IH.C2.-H), 7-58
(s,lH,C2-H), 8-82 (s.br.lH.NH).
N6,5'-O-Dlacetyl-8,2'-S-cycloadenosine
The triacetyl compound (1 -0 g) was dissolved in 50% aq pyridine (10 ml) and
cooled to 0°.
To the solution IN NaOH (10 ml, ca, 2 equiv) was added and it was kept
at 0° for 5 min.
The reaction mixture was neutralized with IN HCl, evaporated in
vacuo, and the residue was dissolved in pyrldine.
Insoluble material was filtered off,
the filtrate was evaporated to give a residue, which was recrystallized from MeOH.
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Nucleic Acids Research
Colorless needles, m. p. 137-138°, were obtained in a yield of 560 mg (64%).
Calcd. forC 14 H 15 N5S.H2O: C, 43-86; H, 4-47; N, 18-27.
H, 4-63; N, 18-23.
Anal.
Found: C, 43-94;
NMR: 1-88 (s, 3H, 5'-OAc), 2-22 (s, 3H, NHAc), 4-10
(d, 2H, 5'-H 2 ), 3-90-4-50 (m,2H,C3-H and C 4 -H), 4-91 (dd, lH,C 2 -H,J 2 ._ 3 -= 2-4
Hz), 6-05(d,lH,C 3 -OH,JoH-3 I H=3-6Hz), 6-63(d,lH,Ci -H L j v .
= 5-2 Hz),
v
8-48 (s, 1H,C2-H), 10-46 (s, br, 1H, NH).
N6-AcetyI-8,2'-S-cycloadenosine
The acetyl compound (560 mg) was dissolved in 50% aq. pyridine (10 ml) and
cooled to 0°.
To the solution, cold IN NaOH(10 ml)was added.
After 5 min at 0°,
the mixture was neutralized with IN HCl, evaporated in vacuo, and the residue was
dissolved in pyridine.
Insoluble material was filtered off andpyridine was evapora-
ted in vacuo to give a residue.
The residue was recrystallized from MeOH to give
colorless needles, m.p. 226-227°, in a yield of 462 mg (93%), Anal.Calcd.: C,
43-46; H, 4-25; N, 21-07.
Found: C, 43-53; H, 4-46; N, 20-93.
NMR:
10-48 (s, NH), 8-49 (s C2-H), 6-59 (d, Cj-H), 5-88 (d,-OH), 4-89 (d-d,C2--H),
4-85 (s, -OH), 4-2-4-6 (m, broad, C 3 .-H), 3-8-4-2 (m, broad,C 4 --H), 3-36 (d, C 5 .-H)
5'-O-Monomethoxytrityl-N6-acetyl-8,2'-S-cycloadenosine
N6-Acetylcycloadenosine (680 mg, 2-1 mmoles) and MMTrCl (780 mg, 2-53
mmoles) were dissolved in pyridine (25 ml).
After keeping the mixture at50°C for
8 hr it was kept at room temperature for further 6 hr.
The reaction mixture was
poured into ice-water containing 2% NH3 and stirred for 30 min.
Crystalline
material was collected by filtration, washed with water, and dried in vacuo over
P2O5.
This material was dissolved in ethyl acetate-MeOH (1:3, vol/vol), treated
with active charcoal, and filtered.
The filtrate was evaporated, a small amount of
pyridine was added, and dissolved in methyl acetate (15 ml).
added dropwise to n-pentane to give an amorphous powder.
The solution was
Yield was 1 -08 g (86-5%).
Analytical sample was recrystallized from EtOH, m.p. 125-128°.
C, 63-79; H, 5-01; N, 12-00; S, 5-49.
S, 5-24.
Anal.Calcd.:
Found: C, 63-35; H, 4-95; N, 11-57;
TLC (CHCl3-EtOH, 10:1), Rf 0-46, NMR: 10-55 (s, NH), 8-56 (s,C 2 -H),
7-20 (s, -C 6 H 5 ), 7-17(s,-C 6 H 5 ), 6-91 (d-d,-C 6 H 5 -OCH 3 ), 6-77 (s, OH), 6-0
(d.Cj.-H), 4-75-5-0 (m,C 2 .-H), 4-0-4-6 (m,C 3 -H,C 4 .-H), 3-7 (s,-OCH3), 2-753-2 (m,C 5 .-H), 2-23 (s
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Nucleic Acids Research
5'-0-Monomethoxytrltyl-8,2'-S-cycloadenoslne 3'-phosphate
S'-MMTr-N^-Acetyl-S^'-S-cycloadenosine (5-95mg, 1 mmole) was dissolved
in pyridine and cyanoethyl phosphate (1-5 mmole) was added.
The solution was
evaporated twice with added pyridine, and finally adjusted to 20 ml.
DCC (1-03 g,
5 mmole) was added and the mixture was kept at room temperature for 13 hr.
Cyanoethyl phosphate (1 >5 mmole) and DCC (0*5 g) were again added and the reaction
was continued for 24 hr.
Water (20 ml) was then added to the mixture and dicyclo-
hexyl urea was filtered off.
The urea was washed twice with 50% pyridine and the
washings were combined with the filtrate.
After extraction with n-pentane (30 ml x 2),
the solution was concentrated and extracted with n-BuOH (30 ml).
The BuOH solution
was washed with H2O (20 ml, 5 ml x 2) and evaporated with added pyridine.
The
residue was added to n-pentane and the precipitated powder was collected by centrifugation (0-7 g, 88%). The powder (670 mg) was dissolved in pyridine (2 ml) and
3% NH3 aq (50 ml) was added.
perature for 24 hr.
n-BuOH (30 ml).
The slightly turbid solution was kept at room tem-
Unreacted material was extracted from the solution with
Further extraction of the water layer with n-BuOH (30 ml x 2) and
evaporation of the BuOH solution with added pyridine gave a residue.
was dissolved in pyridine and added dropwise to n-pentane.
powder was collected by centrifugation.
The residue
The precipitated white
The yield of MMTr-8,2'-S-cycloadenosine
3'-phosphate ammonium salt was 434 mg (78%).
Properties are listed in Table I.
8,2'-S-Cycloadenosine 3'-phosphate
MMTrAsp (10 mg) was dissolved in 80% AcOH (1 ml) and kept at room temperature for 1 hr.
After evaporation of AcOH in vacuo, the residue was dissolved in 50%
aq pyridine and applied to paper chromatography in solvent B.
A s p was extracted
from the band migrating at Rf 0*1. When this material (5 OD275) was incubated with
crude snake venom (10 mg/ml, lOjol) in 1M NH4HCO3 (pH 9*1, 20/ul) at 37° for 14 hr
liberation of Pi was not observed.
The incubation of 8,2'-S-cycloadenosine 5'-phosphate
in the same condition gave 50% dephosphorylation.
Thus, the position of the phosphate
group was confirmed to be at the 3'.
8,5'-Cycloadenosine 2',3'-cyclic phosphate
8,5'-S-Cycloadenosine8 (281 mg, 1 mmole) was dissolved in DMF (20 ml).
To
the solution, cyanoethyl phosphate (4 mmoles/5 ml pyridine) (2-5 ml, 2 mmoles) and
DCC (2-06 g, 10 mmoles) were added.
for 2 days.
1354
The mixture was kept at room temperature
The reaction mixture was poured into 50% aq pyridine containing tri-
Nucleic Acids Research
ethylamlne(0-56 ml, 4 equiv).
A small amount of n-pentane was added to the mixture
and dicyclohexylurea was filtered off.
The filtrate was extracted with n-pentane
(20 ml x 3) and the water layer was applied to a column (3-1 x 41 cm) of DBAE-cellulose (bicarbonate form).
The column was eluted with 0-2M triethylammonium
bicarbonate (3 1) and H2O (3 1) in a linear gradient.
2',3'-Cyclic phosphate was
eluted in the third peak in a yield of 11,500 OD284 units (64%).
Properties were as
listed in Table I.
8, 5'-S-Cycloadenosine 3'-phosphate
8,5'-S-Cycloadenosine 2',3'-cyclic phosphate (3,500 OD284 units) RNase M
(2-5 mg/ml) in 1 ml, 1M NH4OAC (pH 5-0) 10 ml and H2O 40 ml were incubated at
37° for 2 days.
The mixture was concentrated to half its volume and added to EtOH
containing CaCl2*2H2O (160 mg).
dissolved in H2O (10 ml).
Precipitates were collected by centrifugation and
EtOH was added to cause precipitation of the Ca salt.
This material was suspended in 50% pyridine and Dowex 50 x 2 (pyridinium) resin
was added.
The solution was applied to a column (1 -3 x 10 cm) of Dowex 50X
2 (pyridinium) resin to convert it to the pyridinium salt.
Yield was 2,900 OD234
units (80%).
N6-2'-Dibenzoyl-8,5'-S-cycloadenosine 3'-phosphate
8,5'-S-Cycloadenosine S'-phosphate (4,700 OD285. 0-262 mmole) was passed
through a column of Dowex 50 x 2 (20 ml, pyridine form) and the eluant was evaporated.
To the residua, tetraethylammonium benzoate (made from benzoic acid
320 mg, 2*62 mmoles) was added and the total mixture was evaporated with added
toluene three times to give a glassy residue.
Benzoic anhydride (960 mg, 4-24
mmoles) was added to the residue and the reaction mixture was heated at 50° to
give a homogeneous solution, which was kept at 25-28° for 5 days.
50% Pyridine
was added to the reaction mixture, which was extracted with n-pentane (30 ml x 2)
and CHCI3 (30 ml x 20 ml).
CHCI3 was evaporated in vacuo and the residue was
evaporated with added pyridine and redissolved in pyridine.
Precipitates were
obtained by adding the pyridine solution to ether-pentane (3:2).
Yield was 310 mg.
This material was dissolved in pyridine (5 ml) and AC2O (2-5 ml) was added.
After keeping the reaction mixture at room temperature for 15 hr, it was evaporated to give a residue.
The residue was dissolved in 50% pyridine (50 ml) and
applied to a column (1-3 x 20 cm) of Dowex 50 x 2 (pyridine form) resin.
Eluants
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Nucleic Acids Research
were kept at room temperature for 2 hr, coevaporated with pyridine, and added to
ether-n-pentane (3:2) to cause precipitation.
Precipitates were collected by centri-
fugation and dried in vacuo over P2O5. Yield was 110 mg (3,700 OD30o nm).
2',5'-Di-Q-Acetyl-8-bromoadenosine 3'-phosphate
Pyridlnium BrAp (5,000 OD260« ° ' 3 3 nunole) was dissolved in H2O (3 ml) and
pyridine (15 ml).
AC2O (15 ml) was added with cooling and stirring.
was kept at room temperature for 2 hrs.
The mixture
The reaction mixture was evaporated in
vacuo then the residue was dissolved in 50% pyridine and applied to paper chromatography in solvent F.
Bands having Rfs 0-70, 0-49 and 0-37 were detected.
The
band having Rf 0-49 was eluted with 50% pyridine and the extract was applied to a
column (l«l x 15 cm) of Dowex 50 x 2 (pyridinium form).
The pyridinium 3'-phos-
phate was obtained by evaporation of the eluants and precipitation from its pyridine
solution with ether-pentane (3:2).
The powder was dried in vacuo over P2O5 to give
3,200 OD 263 (64%) of 2',5'-diacetyl-BrAp pyridinium salt.
N6-2',5'-O-Tribenzoyl-8-oxyadenosine 3'-phosphate
8-Bromoadenosine 3'-phosphate (pyridinium salt, 13,800 OD263. 0-92 mmole)
was dissolved in 25% pyridine (150 ml) and applied to a column (1-1 x 41 cm) of Dowex
50 x 2 (pyridinium).
Elution with 25% pyridine (150 ml) and evaporation of the
eluants gave a residue.
Tetraethylammonium benzoate (9*2 mmoles) were added to
the residue and the solution was evaporated three times with added pyridine.
residue was finally evaporated with toluene.
The
Benzoic anhydride (2-35 g, 18*4 mmoles)
was added, the mixture was heated at 50°, to obtain a homogeneous solution, then
kept at room temperature for 7 days. The reaction was stopped by adding 50% pyridine (50 ml) and the solution was extracted with n-pentane (30 ml x 3) andCHCl3 (30 ml,
20 ml x 2).
The CHCI3 layer was washed with H2O (10 ml) and evaporated in vacuo.
The residue was evaporated with pyridine and precipitated in ether-pentane (3:2).
Precipitates were collected by centrifugation and dried over P2O5. This material
was dissolved in AC2O (10 ml) and pyridine (10 ml) and kept at room temperature for
15 hr.
The reaction mixture was evaporated and the residue was dissolved in 50%
pyridine (100 ml).
The solution was applied to a column (1 -1 x 39 cm) of Dowex
50 x 2 (pyridinium form) and eluted with 50% pyridine.
kept at room temperature for 2 hr.
Eluants were combined and
The solution was evaporated in vacuo with added
pyridine and precipitates were obtained with ether-n-pentane (3:2).
1356
Precipitates
Nucleic Acids Research
were dried in vacuo over P2O5. A small amount of this material was deacylated with
metbanolic ammonia at room temperature for 20 hr.
The resulting 8-oxyadenosine
3'-phosphate was identical with an authentic sample by paper chromatography in solvent B.
5'-O-Monomethoxytritylformycin
Formycin (0*5 g, 2-06 mmoles) was dissolved in pyridine (50 ml) and MMTrCl
(0-7 g, 2-27 mmoles) was added.
for 20 hr.
The reaction mixture was poured into ice-water (300 ml) containing 0-5%
NH3, with stirring.
CHCI3.
The mixture was stirred at room temperature
After addition of NaCl (30 g), the nucleotide was extracted with
The CHCI3 layer was evaporated and MeOH was added to the residue.
Evaporation of MeOH gave a white powder in a yield of 0-335 g (89%). TLC (CHCI3EtOH, 5 : l) Rf 0-51.
t^-Dimethylaminomethylene-S'-O-MMTr-formycin 2' t 3'-cyclic phosphate
MMTr-Formycin (242 ing, 0-5 mmmole) was made anhydrous by evaporation
three times with pyridine and finally with DMF. The residue was dissolved in DMF
(50 ml) and cyanoethyl-phosphate (1-5 mmole) and DCC (515 mg, 2-5 mmoles) were
added.
The mixture was kept at room temperature for 20 hr.
The reaction was
stopped by adding 50% pyridine (20 ml) containing Et3N (0*63 ml, 4*5 mmoles).
Dicyclohexylurea was filtered off and the filtrate was extracted with n-pentane
(1 -5 ml x 3).
The aqueous solution was evaporated in vacuo and the residue was
dissolved in n-BuOH (30 ml) and H2O (10 ml). The BuOH layer was washed with
H2O (3 ml x 4) to remove cyanoethyl-phosphate and Inorganic phosphate.
BuOH
was evaporated in vacuo and the residue was evaporated three times with pyridine.
Precipitation in ether-pentane (3:2) gave a powder, which was collected by centrlfugation and dried in vacuo over P2O5. Yield was 0-347 g (92%).
Formycin 2',3'-cyclic phosphate
N°-Dimethylaminomethylene-5'-MMTr-Fp (347 mg, 0-46 mmole) was dissolved in 80% AcOH (100 ml) and kept at room temperature for 2 hr. The solvent
was evaporated and traces of AcOH were removed by coevaporation twice with H2O.
The residue was evaporated three times with pyridine and finally dissolved in
pyridine (3 ml).
Addition of the solution to n-pentane-ether (2:3) gave precipitates,
which were collected by decantation.
To the powder, methanolic ammonia (50 ml)
was added and the solution was kept at room temperature for 24 hr.
MeOH was
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Nucleic Acids Research
evaporated in vacuo, the residue was dissolved in H2O (200 ml) and applied to a column
(2-1 x 54 cm) of DBAB-cellulose.
After washing with H2O (1-5 1) the nucleotide was
eluted with a linear gradient of 0-2M tetraethylammonium bicarbonate (2 I) and H2O
(2 1).
Formycln 2\3'-cyclic phosphate was obtained in the third peak (592 OD295) and
formycin 2'(or 3') phosphate was obtained hi the fifth peak (1,760 OD295>«
Formycln 2'
(or 3') phosphate from the fifth peak (1,760 OD295, 0-17 mmole) was passed through a
column (1 -2 x 30 cm) of Dowex 50 x 2 (pyridinium form) with elution by 50% pyridine
(150 ml).
Eluants were evaporated, morpholino-di-cyclohexylcarboxamidine (100 mg,
0-34 mmole) was added, and the mixture evaporated three times with added pyridine.
The residue was dissolved in pyridine (20 ml) and DCC (175 mg, 0*85 mmole) was
added.
The mixture was kept at room temperature for 24 hr and evaporated in vacuo.
To the residue, 50% pyridine (30 ml) was added, precipitated dlcyclohexylurea was
filtered, and the filtrate was extracted with n-pentane (20 ml x 3).
The water-layer
was evaporated, coevaporated with pyridine three times and poured into ether-n-psntane
(3:2).
Precipitates were collected by centrifugation and dried over P2O5.
Formycin
2',3'-cyclic phosphate was obtained in a yield of 132 mg, (1,630 OD295. 93%).
Formycin 3'-phosphate
Formycin 2 f ,3'-cyclic phosphate (2,100 OD295, 0*2 mmole) was dissolved in H2O
(30 ml) and 1M AcONH4 (pH 6-0, 6 ml) and RNase T 2 (51 mg) were added.
ture was incubated for 8 hr.
The mix-
The reaction mixture was evaporated in vacuo and the
residue was dissolved in H2O (20 ml). The solution was added dropwise in EtOH
(50 ml) containing CuCl2-2H2O (130 mg, 0-88 mmole).
Resulting precipitates were
collected by centrifugation, the supernatant decanted, and the residue was dissolved
again in H2O (20 ml).
Precipitates obtained upon adding the solution to EtOH (50 ml)
were collected by centrifugation.
This process was repeated three times.
The pre-
cipitates were dissolved in H2O by adding Dowex 50 x 2 (pyridinium form) and absorbed on an active charcoal column (1-3x10 cm).
Washing with H2O (2 ml) and with
50% EtOH containing 5% NH3 gave eluants, which were evaporated to give a residue.
The residue was dissolved in 25% pyridine (50 ml) passed through a column (1 -1 x
34 cm) of Dowex 50 x 2 (pyridinium form).
Elution with 25% pyridine (150 ml) and
evaporation of the eluants gave pyridinium formycin 3f-phosphate hi a yield of 1,500
OD
295 (70%).
1358
Properties are lis.tsed in Table I.
Nucleic Acids Research
N6-2>,5'-Tribenzoylformycin 3'-phosphate
Formycin 3'-phosphate (1,500 OD295, 0-15 mmole) was dissolved in 50%
pyridine (5 ml) and tetraethylammonium benzoate (1-5 mmole) was added.
The
solution was evaporated three times with added pyridine and finally with toluene
three times.
To the residue, benzoic anhydride (680 mg, 3 mmoles) was added
and heated at 45° for a while to obtain a homogeneous solution, which was then left
at room temperature for 5 days.
The reaction was stopped by adding cold 50%
pyridine (30 ml) and the solution was extracted with n-pentane (20 ml x 3) and then
with CHCI3 (30 ml, 20 ml x 2).
evaporated to a residue.
The CHCI3 layer was washed with H2O (5 ml) and
The residue was dried by evaporation three times with
added pyridine and poured into ether-n-pentane (3:2) to cause precipitation.
The
precipitates were collected by centrifugation and dried over P2O5. The powder
was dissolved in pyridine (3 ml) and AC2O (1 -5 ml) and kept at room temperature
for 15 hr.
AC2O was evaporated, the residue was dissolved in 50% pyridine
(50 ml), and passed through a column of Dowex 50 x 2 (pyridine form).
Eluants
were kept at room temperature for 2 hr, evaporated and dried by evaporation with
added pyridine.
The residue was obtained as a powder by precipitation from ether-
n-pentane (3:2).
Precipitates were collected by centrifugation, the supernatant
was decanted, and dried over P2O5. Yield was 0*140 g.
General procedure for synthesis of XpUpG
Properly protected 3'-phosphate (amount listed in Table I) and U(OBz)pG1Bu(iBu)2 (amount In Table II) were separately dissolved in 50% pyridine and passed
through a column of Dowex 50 x 2 (pyridinium form).
Eluants were evaporated in
vacuo and made anhydrous by evaporation with pyridine. Each residue was dissolved in pyridine and poured into ether-pentane (3:2) to give precipitates.
Each
precipitate was dried over P2O5 in vacuo, dissolved in pyridine, combined and
evaporated three times with added pyridine.
Finally the residue was dissolved in
pyridine (10 ml) and concentrated to 1-2 ml.
DCC (amount listed in Table II) was
added and the reaction mixture was kept at room temperature for 2 days with exclusion of moisture.
The reaction mixture was poured into cold 50% pyridine with
cooling, kept at room temperature for 30 min, and precipitated dicyclohexylurea
was filtered off.
The urea was washed with 50% pyridine, washings and filtrate
were combined and extracted with n-pentane (20 ml x 2).
The water layer was
evaporated to dryness, with added pyridine, dissolved in pyridine and poured into
1359
Nucleic Acids Research
P r o p e r t i e s of D i f f e r e n t
Table I
Nucleoiides
x(nn )
Compound
PPC(Rf)
(A)
8.5--5-Ap>
0.32
(B)
0.30
PEP
Km
1.00
B.5--S-AP
0.22
0.12
1.80
8.5--S-4pBz2
0.46
0.51
1.40
8,S'-S-Ap-pyro
0.30
0.20
1.38
8,5'-S-ApUpG
0.09
0.08
1.12
B,2'-S-AAC 4
0.68
8,2'-S-AAc3
0.51
256
225
287.5 295
8,2'-S-AAc2
0.28
8,2'-S-AAc
0.07
231
256
287.5 295
257
232
289.5 297
HMTrA8p-CEP
0.85
0.76
0.07
232
291
299
s
rWrA p
ASp
0.60
0.24
0.68
0.12
1.22
244
277
1.95
276.5
A*pUpO
UpG
0.07
0.10
1.22
220
0.25
0. 25
1.00
Compound
Formycin
PPC PPC
(A) (B>
0.09
5 ' -O-MMTr-Fonnycin
0.51
N6-DHM- 5 ' -O-MMTrFp
0.91
Fp
OH
H2O
284
293
286
296
235
276
284
292
276
284
294
225
277
284
293
301
234
251
301
228
279
232
287
296
320
236
285
277.5 237
297
285
278
294
278
294
295
261
272
283
293
268
280
292
271
281
291
223
293
255
231.5 297
289
290
232
298
289
230
297
244
307
244
307
243
307
243.
304
233
301
23B
267
228
267
235
252
232
297
231
290
277
290
299
315
278
277
277
266
PEP
Rra
UV s p e c t r a
Neutral
229(s) ,287(s) ,294.5
304<s) ,318(8)
2301s) ,2851s) ,295
305(8) .31918)
0.80 270(8) .333
213
269
270
nm)
max 1
OH"
H
234 Is) , 296,3051s) 2341s) , 2 6 0 ( 8 ) 3 0 3 . 5
A.
,304
230
296
05(s)
300 s , 3 4 2 . 5
0.51 0 47 0.85 2301s) , 2 8 6 ( s ) .294.5
305(8) .316
2341s
316(s
268(8) , 2 9 6 . 5 , 3 4 1
296,305(8)
234.5( s ) , 3 0 4
0.81 0.75 1.28
l50%EtOH)234ls)320
2 3 4 ( s ) . 319
235(8) ,343
BrAp
0.22 0.17
265
264
266
2',5'-O-dlAcBrAp
0.52 0.33 1.7
210(s) ,265
263 5
266
266
263 5
BrApBz.,
1.36 234,288
0.80
N6-BzHOAp
0.23 2 . 0
230(8) ,308
234 288
<50lEtOH)231( 8) ,299
2 3 1 1 s ) , 299
228(s) ,317
270
2 6 5 2751s)
2B0
232 298
231.2651s).318
KOAp
0.16 0.08 2 . 0
HOApBz^
0.74 0.63 1.41 2 3 1 . 5 , 298
FpUpG
260
291 m i JU r m ;
£jr\z»j
BrApUpG
3031s)
0.10 0.03 1.22 261
262
263
HOApUpG
0.11 0.04 1.41 260
262
269
Table 1
B
A UG
3'-Mononucleotide a
12Omg,317O OD, 7?
(0.169 mmole)
-AUG
BrAUG
HOAUG
Synthesis of
Dinucleotide
100 mg
(0.121
71 mg. 2400 OD 300
AUG Analogs.
DCC
590
348 ing
ralo]
rag.2470 OD260
yield
2 ml
2 days
1570
2 ml
2 days
600
OD
1 ml
3 days
398
° D 36O
1 ml
3 days
322 OD
260
1 ml
3 days
213
°°260
(1.69 nraole)
227 Bg
(0.11 8mole)
(0.091 mnole)
1200 OC
100 mg,2710 OD26 „ 490 mg.
(0.19 sanole)
(0.103 mmole)
750 ng 1710 OD
Pyridin s
260
(1.1 mole)
(2.39 n n o l e )
286
( 1 . 34 a a o l e )
TOG
100 mg. 1440 O D j 2 0
90 mg.2460 OD26Q
140 mg
(0.136 B » l e )
(0.093 saole)
(0.68 mole)
OD
a.
H u c l closide 3 '-phosphates vere properly protected as described in Experim e n t a l .
b.
Dinucleotide was U(OBz)pGi&u(OiBu)2.
1360
250
Nucleic Acids Research
Table I I I
U l t r a v i o l e t Absorption Properties of AUG analogs
Compound
8,2'-O-AUG
8,5'-O-AUG
8,2'-S-AUG
8,S'-S-AUG
Br-AUG
Oxy-AUG
PUG
AUG
A Qax({ per c o l e of base residue)
pa 7
pH 12
257.5(11300)
258.5(10100)
258.5(11100)
261.5(10600)
218(sh)(7200)
269(9300)
265(b)(11000)
243(sh)(6800)
237(sh)(6800)
260(sh)(9000)
274(b)(9200)
273(b)(9300)
284(sh)(7600)
294(sh)(4600)
294(sh)(4300)
260(11100)
264(10700)
259(10000)
269(9000)
236(sh)(4B00)
234(sh)(8600)
257 5(8000)
261(6800)
293(sh)(3800)
304(2600)
305(sh!(2400)
2 58(11400)
260(10200)
258.5(11700)
pH 2
259(10800}
260-5(11200)
266(11000)
272(10900)
265(Bh)(9600)
275(10300)
282(sh)(9700)
293<sh)(6500)
262(11600)
261.5(9B00)
258-5(8100)
292(sh)(4700)
(sh) means shoulder
(b) means broad
Enzymatic Hydrolysis of AUG Analogs
XUG(OD,,ft)Enzyme(fd)
260
Buffer (/>1)
ASUG
3.0
SVP a (lmg/ml)
10
8
AUG
3.0
BrAUG
3.5
3.5
BNaseMt 2.5mg/ml)lMNH 4 OAc(pH5.0)
10
10
RNaseM(2.5mg/Dl)lMNH4OAc(pH5 0)
SVP(5rag/ral)
lMNH^CO3(pH9.1)
HOAUG
3.0
SVP(5 mg?ml)
2
PUG
3 .6
3.6
1KNH 4 HCO 3 (pH9.1)
20
Temp Time Ratio
(c°) (hr) X:U G
37 4
1.0:1.0-1.0
37
4
1.0:0.95:1.04
37
37
3
3
1.0.0.96:1.03
1.03:1.0:1.1
37
3
0.90:1.0:1.1
RHaseHf 2 . Sag/ml) 2HNH4OAc (pH5. 4)
10
5
37
3
(not measured)
SVP(5jag/ml)
2
37
3
1.05:1.0:0.99
lMNH4HCO3(f
10
lHNH4HCO3(pH9.1)
10
a. SVP stands for snake venom phosphodiesterase.
Hyperchroraicity and tiypochromicity of AUG Analogs
Compound
Wave
length( nm ) Hyperchroraicity(%)
Hypochromicity(%)
8,2'-O-AUG
257
7.7
7.3
8,5'-O-AUG
258
13.6
12.1
8,2'-S-AUG
260
8,5'-S-AUG
280(ma x) 18.7
15.7
260
2.09
U.5
284(ma x) 15.7
13.5
260
6.70
262
7.80
259
4.61
270
9.97
275(ma x) 12.6
PUG
258
6.86
280 (ma x) 17.0
AUG
2.02
13.0
270(ma x) 12.4
Oxy-AUG
2.86
12.1
276
Br-AUG
2.95
13.7
272
6.28
7.24
11.0
4.41
9.09
11.2
6.42
14*. 5
29S
14.9
13.0
258
9.0
8.3
1361
Nucleic Acids Research
ether-n-pentane (3:2).
dried in vacuo.
Resulting precipitates were collected by centrifugation and
The powder was dissolved In 80% AcOH (50 ml) and kept at room
temperature for 1 hr.
and evaporated.
The solvent was evaporated In vacuo, H2O (10 ml) was added
The residues were dried by evaporation with pyridine and poured
into ether-n-pentane (3:2) to give precipitates, which were dried In vacuo.
The pow-
der was dissolved in 9N methanollc ammonia (50 ml) and kept at room temperature
for 14 hr.
MeOH was evaporated carefully, the residue was dissolved In 50% pyri-
dine and diluted with H2O (100-200 ml).
a column of DEAE-cellulose.
After filtration the solution was applied to
Linear gradient elution using 0-2M trlethylammonium
bicarbonate and water gave trinucleotides in appropriate fractions.
the DEAE-cellulose column chromatography was repeated.
If necessary,
Trinucleotides thus
obtained were further purified by Sephadex G10 or G15 gel filtration using 0-01M
trlethylammonlum bicarbonate buffer as the eluting solvent.
Yields are summari-
zed in Table II and properties are summarized in Tables I and III.
General procedure for enzymatic hydrolysis of trinucleotides
(i) Snake venom phosphodiesterase.
The reaction mixture (100 (jl) containing
trinucleotide (3-4 OD units), snake venom phosphodiesterase (1 mg/ml) 10 \il and
1M NH4HCO3 (pH 9-1) 20 |jl was Incubated at 37° for 4 hr.
trated and applied to paper chromatography in solvent F.
The mixture was concenSpots corresponding to
nucleoside and 5-phosphates were cut out and eluted with water (2 ml).
tion was measured and contents of nucleotides were calculated.
in Table IV.
UV absorp-
Results are listed
(11) RNase M: the reaction mixture (100 pi) containing trinucleotide
(3-4 OD units), 1M ACONH4 (pH 5-0) 10 \A and RNase M (2-5 mg/ml) 10 pi was incubated at 37° for 4 hr.
Results are listed in Table IV.
Hyper- and hypochromiclty of trinucleotides
These values were obtained by the comparison of untreated solution and the
incubation mixture of enzymatic digestion, which was diluted with 0-1M KF and 0-05
phosphate buffer (pH 7-0) to 2 ml.
Results are summarized in Table V.
Acknowledgement
A part of this research was supported by Grant-in-Aid for Scientific
Research from the Ministry of Eduction, to which authors gratefully thank.
REFERENCES
(1)
1362
Part XXX: Uesugl, S., Tezuka, T., and Ikehara, M., In preparation.
Nucleic Acids Research
(2) Itehara, M., Nagura, T., and Ohlsuka, B. (1974) Chem. Pharm. Bull., 22,
123
(3) Ikehara, M., Nagura, T., and Ohtsuka, E. (1974) Chem.Pharm.Bull., 22,
2578
(4) Lucas -Lenard, J., and Llpmann, F . (1971) Ann. Rev. Bloc hem., 40, 409
(5) Nlrenberg, M., and Leder, P. (1964) Science, 145, 1399
(6) Donohue, J., and Trublood, K.N. (1960) J.Mol.Blol.. 2, 363
(7) Ikehara, M., and Kaneko, M. (1970) Tetrahedron, 26, 4251
(8) Ikehara, M., Kaneko, M., and Saga 1, M. (1970) Tetrahedron, 26, 5757
(9) Tomita, K., Tanaka, T., Yoneda, M., Fujlwara, T., and Ikehara, M.
(1972) Acta Cryst., A28, 45
(10) Ikehara, M., and Uesugl, S. (1969) Chem.Pharm.Bull., 17, 348
(11) Ikehara, M., Tada, H., and Kaneko, M. (1968)
Tetrahedron, 24, 3487
(12) Travale, S.S., and Sobell, M. (1970) J.Mol.Biol., 48, 109
(13) Ikehara, M., Uesugl, S., and Yoshlda, K.
(1972) Biochemistry, 11, 830
(14) Koyama, G., Maeda, K., Umezawa, H., and Iltaka, Y. (1966) Tetrahed.
Letters, 597
(15) Pruslner, P., Brenner, T., and Sundarallngam, M. (1973) Biochemistry,
12, 1196
(16) Tener, G.M. (1961) J.Amer.Chem.Soc., 83, 159
(17) Ikehara, M., and Uesugl, S. (1972) Tetrahedron, 28, 3687
(18) Irie, M. (1967) J. Bloc hem., 62, 509
(19) Rammler, D.H., Lapldot, Y., and Khorana, H.G. (1963)
85; 1989
J.Amer.Soc.,
(20) Sawa, T., Fukugawa, Y., Homma, Y., Takeuchl, I., and Umezawa, H.
(1967) J.Antibiotics, 20A, 317
(21) Naoi-Tada, M., Sato-Asano, K., and Egaml, F. (1959) J. Bloc hem., 46,
757
(22) Uesugi, S., Nagura, T., Ohtsuka, E., and Ikehara, M., in preparation
(23) UV absorption spectra were taken with a Hitachi EPS-3T or 124 spectrophotometer.
In Fig. 1-5 solid line shows the spectrum taken at pH 7-0,
dotted line at pH 2-0 and broken line at pH 12-0.
Paper chromatography
was performed on Toyo filter paper No. 51A in the following solvent systems:
(A), EtOH-lM NH4OAc (7:3); (B), 1-PrOH: NH4OH:H2O (7:1:2).
Paper
electrophoresis was performed in O'OIM triethylammonium bicarbonate
(pH 7*5) at 35 V/cm.
Rm stands for a migration ratio relative to adeno-
sine 2',3'-cyclic phosphate (Ap>).
1363
Nucleic Acids Research
1364