Aminonucleosides and their derivatives. FVr Synthesis of the 3

Volume 6 Number 2 February 1979 Nucleic Acids Research

Aminonucleosides and their derivatives. FVr Synthesis of the 3'-amino-3'-deoxynucteoside 5'-phosphates

A.V.Azhayev, A.M.Ozols, A.S.Bushnev*, N.B.Dyatlcina, S.V.Kochetkova, L.S.Victorova,M.K.Kukhanova, A.A.Krayevsky and B.P.Gottikh

Institute of Molecular Biology, USSR Academy of Sciences, Vavilov St. 32, Moscow 117984, and*Moscow Institute of Fine Chemical Technology, M.Pirogovskaya St. 1, Moscow 119435, USSR

Received 5 January 1979

.ABSTRACTA new procedure hes been developed f o r the s y n t h e s i s of

3 ' -8mino-3 ' -deoxyr ibonucleos ides of adenine , cy tos ine enduracil by condensing the trimethylsilylated bases with per-acylated 3—ezido—3—deoxyribose derivative. The ezido groupcould subsequently be reduced to amino. The 5'-Phosphates ofthese nucleosides have been prepared and the aneloguee havebeen tested for their ability to stimulate the ribosome-ce-talyzed reaction of 3'(2f)-O-(N-formylmethionyl)edenosine5'-phosphate with phenylslenyl-tENA.

INTRODUCTION

Nucleosides, in which one of the hydrosyl groups of the

sugar residue is substituted by the amino function, ere of

interest to present-day molecular biologists and enzymolo—

gists.

Introduction of the 3'-8niino-3l-deoxy- end the 2l-ami-

no-2'-deoxyedenosine into the tRNA 3'-'tenninus was among the

methods which enabled the direction of aminoacylation of

different tRNA types to be determined . The fj'-triphosphat-

es of the 2 1- and the 3'-sminonucleosides appeared to be ef-

fective inhibitors of the DNA-dependent RNA-polymerese from

B.coli . Various 3'-aniino-3l-deox3'nucleosides may also be

used as a tool for the study of the peptidyltransferase cen-

ter of ribosomes.

The amino acid derivatives of the 3f-amino-3'-deoxyade-

nosine bound to the acceptor site of the peptidyltransferase

center are known to act as inhibitors of protein synthesis .

The results obtained indicated that the acyl-amino acid deri-

vatives of 3'-eniiQO-3l-deoxyadenosine 5'-phosphate bind to

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Nucleic Acids Research

the donor s i t e of the ribosomal pept idyl transferase and thusinhib i t the t ransfer of the acylsmino acid residue fromCACCA-(AcLeu) to the phenylalanyl-tRNA .

ftith these fac ts in mind i t was f e l t to be important todevelop a procedure which would enable subs tan t ia l amountsof the 3 l-amino-3'-deoxynucleosides and the i r 5'-phosphatesto be prepared.

KbSULTS AKD DISCUSSION

Ear l i e r we have reported the synthesis of the S'-ami-110--3 '-deoxyuridine obtained by stepwise modification of u r i -dine and also the method for the preparation of 3 '-8mino-3 l--deoxyadenosine ' . In th is paper we wish to report the syn-thes is of the adenine, cytosine and u rac i l containing 3 ' --amino-3'-deoxynucleosides and the i r 5 ' -phosphstes. Theaminonucleosides were obtained by means of glycosidation ofthe s i ly la ted N-benzoyladenine, cytosine and uracil e i therwith the e a r l i e r reported 3-acetemido-3-deoxy-1,2-di-O-ace-tyl-5-O-p-nitrobenzoyl-B-D-ribofuranose and the subsequentremoval of the protect ive groups, or with the 3-szido-3-deo-xy-1,2-di-O-acetyl-5-O-p-nitrobenzoyl-B-D-ribofuranose andthe subsequent reduction of the azido group (Scheme 1) .

Synthesis of VII via azidonucleoside has been achievedby us before. However, we would l ike to describe here asimplified procedure for obtaining VII , and also the synthe-s i s of VIII and X via appropriate azidonucleosides.

The s t a r t i ng mateiial foi the synthesis of I I was 1,2--O-isopropylidene-3—O-p-toluenesulfonyl-B—D-xylofuranose(XV) prepared from 1,2-O-isopropylidene-co-D-xylofuranose(XI). 1,2:5,5-l ' i -0-isopropylidene-3-0-p-toluenesulf onyl-oc--D—glucofuranose (XIII) can also serve as a s t a r t i ng materi-a l for the synthesis of XV /7. The fur ther synthesis of I Iwas performed as described in ' with a few modifications(Scheme 2 ) .

The glycosidetion of the s i ly la ted UD-benzoyledenineand cytosine with I was performed in the 1,2-dichloroethane-ace ton i t r i l e mixture in the presence of stannic chloride.Acetemidonucleosides I I I and IV were obtained af ter the gly-

626



H OSiMe,

SiMe,

R'O OAc

X OAo

1 , 1 1

H 0

X OH

III,V,VII

0 > .R- -C-P \S

I.III.IVi 1=

II, V, VI, XT:

VII.VIII.X:

X OH

IX.X

8-HH-O-CH3

X- -H 3

Z= -HH2

R'-

X

IV

?,-c-

OH

,VI,VIII

o

cosidation procedure and the removal of the acyl groups with

methanolic ammonis in a good yield. Removal of the acetyl

group from the aliphatic amino function in III and IV was

observed to proceed under drastic conditions (IfleOHa/MeOH,

A, 24 hr). The yield at that step did not exceed 50% and

considerable darkening of the reaction mixture made the iso-

lation of the desired aminonucleosides difficult.

Thy glycosidation of silylated N -benzoyladenine and

8Z7



Scheme 2

HO

R-

cytosine with II in 1,2-dichloroethane in the presence of

stannic chloride gave (after the removal of ecyl groups)

azides V end VI with an overall yield of 68% and 72.5% from

II, respectively. The reaction of ailyleted urecil with II

in 1,2—dichloroethane in the presence of stannic chloride



gave the mixture of the desired N^-riboside and N^.N^-bis-

riboside, as was obvious from the 1MB spectra of the sepa-

rated products. The formation of bisribosides in the reacti-

on of silylated urecyls with perecylated sugars in 1,2-di-

chloroethane in the presence of stannic chloride was report-10

ed by Vorbriiggen et al. . \/hen the reaction was run in

acetonitrile, the overall yield of ezide IX from II was 97%

(no No—riboside or N^,N^-bisriboside was detected). The re-

duction of the azido group in nucleosides V, VI and IX with

triphenylphosphine in pyridine, followed by treatment of the

reaction mixture with ammonium hydroxide, resulted in amines

VII, VIII and X in a high yield.

The simplicity and high yield of the aminohncleoside

vi8 the azido nucleoside procedure in comparison to the pro-

cedure via the ecetamidonucleoside as well as the advantages

of synthesis of II (against I ) , offer the possibility of ob-

taining larger quantities of sugar in a shorter space of time

and suggests that the route via the azide is more promising.

Another advantage of the azido group is that it may 8Ct as

a completely inert form of the blocked amino function end

can be easily transformed to the amino group in the nucleoti-

de or oligonucleotide structures .

Hereafter we shall report the procedure for the synthe-

sis of 3'-emino—3'-deoxynucleoside 5'-phosphates starting

from azides V, VI and IX (Scheme 3). Phosphoryletion of V, VI

and IX was achieved with phosphorus oxychloride in triethyl-

phosphate, following a modified procedure . Reduction of

the azidonucleotides XXII-XXIV with triphenylphosphine in

pyridine in the presence of ammonium hydroxide resulted in

Scheme 3

OH

XXII -XXIV XXV-XXVII

X X I I : B = A d a ; XXV : B = A d e ;

XXIII : B = Cyt; XXVI: B = Cyt;

XXIV : B = Ura; XXVII: B = Ura.

029



the production of aminonucleotides XXV-XXVII in nigh y ie ld .The spec t ra l , chrometographic end electrophoret ic characte-r i s t i c s of the obtained nucleosides and nucleotides are givenin Table I .

Table 2 i l l u s t r a t e s the PMR spectre of the obtained nuc-leosides and nucleot ides . The spectra of V and VII are simi-l a r to those reported in . The value J ^ , 2 i decreases inthe sequence azide-amine-8cetamide. This may indicate theshi f t of the conformations! N^=i S equilibrium of the ribofu-

11ranoae cycle to the N-region in th i s sequence J.As i t was reported ear l ie r the cytidine and i t s 5 '-phos-

phate promote the riboeome catalyzed reac t ion of the 3'-tev-minel fragment of the peptidyl-tRNA-3'(2')-O-(N-f ormylinethio-

14 1"Snyl)edenosine 5'-phosphate with the phenylalanyl-tRNA ' ' .

TABLE 1SPECTRAL* CHROMATOGRAPHIC AND ELECTROPHOkETIC

CHARACTERISTICS OP THE OBTAINED NUCLEOSIDES AND NUCLEOTIDES

Compound :

I I I

V

V I I

XXIIXXV

IV

VI

VIIIXXIII

XXVI

IX

X

XXIV

XXVII

\pH 7.0

259(15300)259(15200)259(15100)259(15570)259(15740)270(8690)270(8750)270(8960)270(8700)270(8620)261(9830)261(9420)261(9930)261(9610)

: Rf in

A ;0.520.550.33O.360.200.290.440.210.350.15O.63

0.350.380.25

system;B :

0.850.940.710.310.180.680.830.560.200.090.660.460.160.06

pm 2.5.

1.060.911.850.000.881.361.001.950.001.050.001.330.000.14

ppH 7.5pu

-

-

-

0.860.80

-

-

-

1.000.91

-

-

1.081.00

KUV-spectre were recorded i n 0 .1 M Ne-phosphate buf fe r

(pH 7 . 0 ) .

630



TABU 2SH1VTS (ppm) 1HD SFH-SPIB COdFLIHQ O0H31ABT8 (JH») Of THE OMilBBD

SU0LEO3IDE3 AID BDOLSOTIDES*

type

proton

H-8

H-2

H-1'U,, 2 , )H-2'(J2, 3,)H-3 'U 3 , f 4 . )

^ ' ( • V . S ' a . b *H-5'a,b(J4, 5 , a

CHjCO

Type of proton

H-6 (J6 5)H-5 (J5 6 )H-I'U,, 2 , )H-2'(J. , ' 3 , )

1

B-3'(J-,, . , )3 i4'

^' 'V.S'a.b

Type of proton

H-5 'U 4 , c i a b

OH,ci

Type of proton

H-6(J6>5)

H-1'(J^, 2 , )H-2'(J2 , '3 , )H-3-(j 3 . ; 4 . )

^ ' ^ ' . S a . b )H-5'a,b(J4, 5 ,

1

t

.b>

1

)

11

1

7554

4

>,b)3

3

in** '

8.33 a8.08 a

5.9Od(2.0)

+ HODi4.52-3.15 m

2.00 a

IT*.*

7.97d(7.5)6.06d(7.5)5.87d(1.0)

+ HODi

4.95-3.70 a

IV***

2.08

II*.«

.93d(8.0)

.95d(8.0)

.93d(4.0)

.67dd(5.0)+ HODi

.33-4.11 m

.93dd(J4,>5,

•0. J 4 . i 5 . b 4

65443

t1

a

T»* ;

8.47 a8.27 a

.01d(6.0) 6.•11dd(5.5) 4..41dd(5.5).O7dd(3.5) 4 #

.73t(3.5)-

1 " * * 1

7.92d(8.0)5.87d(8.0)5.85d(4.O)

4.45t(4.O)

+ HOD:

4.12-3.91 m

1

3.7Ot(4.O)-

I***

7.99d(8.0)5.93d(8.0)5.89d( 1.0)4.37dd(5.5)3.46dd(9.5)

+ HODi

4.19-3.71 00)

Compound

vu». j8.47 a8.25 a

O3d(3.O) 6.4Odd(5.5)

+ HOUi 5 .

00-3.20 m

-

vm«»

7.88d(7.5)5.93d(7.5)5.81d(1.5)

4.2Odd(5.5)

3.3Odd(8.O)

4.15-3.72 n

[ Tin***

-

| mv*.*8.05d(8.0)6.02d(8.0)5.99d(5.5)4.68t(5.5)

+ HODi

4.44-4.26

4.10 u

VII*** '

8.38 a8.28 a

i6d(3.0)

+ HODi07-3.87 11

-

6.5.

4 .4 .

cm*** * uv***8.58 a 8.38 •8.10 a 7.97 a

03d(5.8) 5.93d(2.8)01dd(5.0) 4.48dd(5.5)

+ HOD 3.14dd(7.3)63-4.33 m 3.06-3.8315u

-

1 mil*** ' nvi»**

8.03d(76.1Od(75.95d(4

4.6Ot(4

+ HOD

4.43-4.

5).5)0)

0)

36

| mii**»

11

H-21 +

7.93d(7.5)6.07d(7.5)5.9Od)2.O)

4.72-4.33 aH-2' + H-4'3.95dd(J3, 2 ,5 .5(

11 Jj , 4,8.O)4.T4-4.33 m H-2'-»

H-4'

; mi***

4.18 u-

urn***8.02d(8.0)5.94d(8.0)5.93d(2.5)

H-4' + HODi 5.1O-4.3Oa3.94t(J3 ,

m H-4' +. J - 7.0)

H-2' + HODi 5.10-4.30m

4.1Od(4.O)

Prior to recording the spectra

Solution In

al l tha oompounda wara twloe 00—«raporat*d with D-0;

a • alnglet, d - doublat, t • tr iplet , dd • doublet of doubleta, m - nultiplet, u - unreaolred.

The analogs of cytidine so obtained were tested for their

stimulating ability. Fig. I demonstrates that VIII end 21-

amino-2'-deoxycytidine ( a kind gift of prof. F.Eckstein)

stimulate the ribosomal process in practically equal measure

and 2-3 times as actively as cytidine. We can give no expla-

631



[stimulator] , mMF i g . 1 . Stimulation of the react ion of 3'(2')-O-(N-formylme-thionyl)edenosine 5'-phosphate with [%]Phe-tENA using: I -cytidine 5'-phosphete; 2 - XXVI; 3 - VIII; 4 - 2 l -emino-2 l -deoxycytidine; 5 -cy t id ine ; 6 - XXIII. The react ion mixturecontained ribosomes 110 pmol, pHJPhe-tENA (3-4).10 cpm(0.7-0.9 pmol); incubation and further treatment was carriedout according to *.

nation for th i s difference so f a r . At the seme time XXVI i seven more effective than cytidine 5 '-Iactive analog, reported innot influence the reac t ion .

14 whereas the azide XXIII does

EXPERIMENTAL16 and I syn-Compound XI was obtained as described in

Q

thesized according to . Thin layer chromatography was runon the "Silufol UV254" p la tes (Kavalier, Czechoslovakia) inthe n-butanol-water-acetic acid (5*3:2, system A), the i s o -propanol-NHÔH-water (7 :1 :2 , system B), the ethylacetate-ben-zene (3 :17 , system C), the ethylacetate-benzene (1 :4 , systemD). The paper electrophoresis was carried out on "F i l t rak )63"(GDE) paper in a 6% acetic acid, pH 2 .5 , or in 0.025 U t r i e t -

63a



hylammonium hydrogen carbonate buffer, pH 7.5. UV spectra were

taken with "Beckman 25" (USA), IE spectra with "UE-10" (GDE).

FMB spectra were registered with "Varian XL-1OO" (USA) in

with tetrsmethylsilane as the internal standard or in

DMSO or DgO with tert-butanol as the internal standard (s =

singlet, d = doublet, t = triplet, dd = doublet of doublets,

dt = doublet cf triplets, m = multiplet, u = unresolved; in

ppm scale).

1,2-0-Isoprbpylidene-3-O-P-toluenesulfony1-5-0-

-benzoyl-w,-D-xylofurenose (XII)

To a ccoled solution (0°) of XI (81 g) in dry pyridine

(120 ml) benzoyl chloride (50 ml) in dry pyridine (70 ml) was

added dropwise and the mixture was kept overnight at 4 .

p-Toluenesulf onyl chloride (165 g) was then 8dded and the

mixture was kept for 4 hr at room temperature. The mixture

was poured onto 350 ml of crushed ice, extracted with chlo-

roform (3 x 200 ml). The extracts were combined, washed with

saturated sodium hydrogen carbonate, dried over NapSO^, eva-

porated to dryness and co-evaporated with toluene (2 x 100 ml)

The residue was dissolved in 50 ml of benzene and fil-

tered through the silica gel column (3 x 20 cm) 8nd the solid

was washed with benzene. The filtrate W8S evaporated to dry-

ness and the residue was crystallized from ethanol. Yield

142 g (73.5%) of XII; m.p. 90-92°; Ef0.58 (chloroform); PME

in CDCly 7.98-7.01 (m, 11 H, H^C^SGg + CgHcCO), 5.96 (d,

1H, J1 24HZ, H-1), 4.93d, 1H, J2 14Hz, J2 ÔHz, H-2), 4.66-

-4.19 U, 3H, H-4 + H-5a,b), 2.26>(s, 3H, H-jC-C^SOg-), 1.49

and 1.3O(2s, 6H, 2CH~). Anal. Calcd. for C22H240gS(448.48):

:58.92%C, 5.39%H; Found: 58.83%C,

1>2-0-Isopropylidene-3-0-p-toluenesulfonyl- ot^D-xylo-

furanose (XV)

To a solution of XII (72 g) in ethanol KOH (14 g) in

water (20 ml) was added. The mixture was boiled for 20 min,

cooled to a room temperature, neutralized with 4% HC1 (pH 7)

and evaporated to dryness. Chloroform (250 ml) and water

633



(250 ml) were then added to the residue. T e organic layer

was separated, end the water layer extracted with chloroform

(2 x 100 ml). The organic extracts were combined, washed

with 10% sodium hydrogen carbonate solution, dried over

N80SG4 and evaporated to dimness. Crystallization of the re-

sidue from hexane-chloroform gave 42 g (76%) of XV; m.p.

83-84°, Rp 0.24 in C; PME in CDCly 7.74 and 7.30 (2d, 4H,

J8Hz, H3C CgH^SOg-), 5.84(d, 1H, <L, 24Hz, H-1), 4.84(d, 1H,

J3>2OHz, J3>43Hz, H-3), 4.53 (d, 1HJ J2)14Hz, J^GHz, H-2),

4.29 (dt, 1H, J4>3 3Hz, J4>5|8>b 8Hz, K U ) , 3.65*(u, 2H,

H-5e,b), 2.40(3/311, H3C-CgH^SO2-), 1.41 and 1.22(2S, 6H,

2CH3). Anal calcd. for C, rH20G,-7S(344.38): 52.32%C, 5.85%H;

Found: 52.28%C, 5.89%H.

112-0-Isopropylidene-3—O-p-toluenesulfonyl-5—0-tetrabydropyranyl- c*-D-xylof uranoae (XVI)

To a solution of XV (25 g) in dry N^N-dimethylformamide

(80 ml), containing 2,3-dihydropyrane (20 ml), a 6% solution

of HC1 in N,N-diinethylf ormamide (8 ml) was added. After 20

min triethylamine (10 ml) was added toa stirred solution, the

mixture was poured into water (200 ml) and extracted with

ether (3 x 100 ml). The extracts were combined, washed with

water (2 x 100 ml), dried over Ne-jSO and evaporated. The

residue was co-evaporeted with pyridine (75 ml) then with

toluene (3 x 75 ml) and finally dried in vacuo to give an oi-

ly residue. Yield 31 g (99%) of diasteriomers XVI, R . 0.55 in

C.

3-Azido-3—deoxy-1,2-0—isopropylidene-cO-D-ribofurenoae

(XIX)

A solution of XVI (29 g) in hexamethylphosphoric triami-

de (150 ml), containing lithium ezide (18 g), was stirred for

3 hrs at 150°. The mixture was cooled to room temperature,

diluted with water (300 ml) end extracted with ether (6 x 200

ml). The combined extracts were washed with water (3 x 150 ml)

and evaporated. The residue was co—evaporated with toluene

(3 x 50 ml), dissolved in chloroform (50 ml) and filtered

through a silica gel column (3 x 10 cm). The silica gel was

634



washed with, chloroform. The filtrate and washings were com-

bined and evaporated to dryness (TLC in D: B~ 0.54- and E~

0.45, presumably XVIII and XVII).

A 75% aqueous acetic acid (300 ml) was then added to the

residue and solution was kept for 15 hrs at room temperature.

The mixture was evaporated, co-evaporated with n-butanol

(2 x 50 ml) end then with toluene. The solution of the resi-

due in system C (10 ml) was applied to the silica gel column

(3 x 30 cm). The elution was run, using the same system. The

fractions, containing pure azide XIX (control-TLC in D), we-

re combined, evaporated and the residue was dried in vacuo.

Yield 7.9 g (54%) of XIX, IL> 0.20 in D; IE: 0 Y CHC1, 2 114

cm (-No); PUR in C D d y 5.76(d, 1H, J1 2 4Hz, H-1), 4.70

(t, 1H, J2 n 4Hz, J2 3 4 Hz, H-2), 4.2O-3.40(m, 4H, H-3 +

+ H-4 + H-5a,b), 1.52 and 1.32 (2s, 6H, 2CH3).

3—Azido—3—daoxy-1,2-O-isopropylidene-5—O—p-nitrobenzoyl-

- ot—D-ribof uranose (XX)

To a solution of XIX (7.5 g) in dry pyridine (70 ml)

8nd 1,2—dichloroethane (30 ml), cooled to 0°, p-nitrobenzoyl-

chloride (7.1 g) was added, the mixture was kept for 1 to at

room temperature and poured onto 300 ml of crushed ice. The

reaction mixture was extracted with chloroform (2 x 200 ml),

the combined extracts were thoroughly washed with the saturat-

ed NaHCOn, dried over Na2SG^,, evaporated and then twice'co-

evaporated with toluene. The syrupy residue W8S filtered

through the silica gel column (3 x 20 cm), the silica gel

W8S washed with chloroform. The filtrate and washing were

combined and evaporated. The residue was crystallized from

ethenol. Yield 9.1 g (71%) of XVIII (needles), m.p. 81-83°,

RP 0.68 in C, IE: $ CHC1, 2114 cm"1 (-NO, PMR in CDC1,:j) j j

8.20(d, 4H, J1, 5 Hz, UpN C^CO-, 5.81(d, 1H, J^ 24Hz,) 3i, 4 .75 ( t , 1H, J 2 14Hz, J 2 34Hz, H-2), 4.67-4.25(m, 3H,

H-4+H-5a,b), 3.32(dd) 1H, J^ 24Hz, J^ ^ OHz, H-3), 1.55 and1.33 (2C, 6H, 2CH3). Anal. Calcd. for'c i 5H16N4Or7(364.3)::49.45%C, 4.43%H, 15.38% N; Found: 49.21%C, 4.52% H, 15.18%N.

3-Azido-3-deoxy-5-O-p-nitrobenzoyl-D-ribofuranoses (XXI)

A suspension of XVIII (9 g) was s t i r r e d in a 7O?5 formic

635



acid (300 ml) for 1.5 hr at 50°. The reaction mixture was

evaporated and then co—evaporated with n-butanol and toluene.

The residue was dried in vacuo. Yield 7.8 g (9&%) of XIX, E f

0.18 in D.

3-Azldo-3-deoxy-1,2-di-0-Bcetyl-5-Q-p-nitrobenzoyl-

-B-D-ribofuranose (II)

Acetic anhydride (90 ml) was added to a solution of XXI

(7.5 g) in dry pyridine (120 ml). The reaction mixture was

kept for 2 hrs at room temperature, poured onto 300 ml of

crushed ice and extracted with chloroform (3 x 150 ml). The

extracts were carefully washed with the saturated aqueous

NaHCGo» dried over Ne2SC4, evaporated and the residue was

twice co-evaporated with toluene. Crystallization of the re-

sidue from chloroform-hexane yielded 8.7 S (89%) of II (need-

les); m.p. 103-104°, Ej 0.67 in D, IE: $mgx CHCl^ 2119 cm"'1

(-N-j), PME in CDCly 8.22(s, 4H, O ^ C ^ C O - ) , 6.12(s, 1H,

C) H-1), 5-35Cd, 1H, J2,1OHz, J2>3 4.5Hz, H-2), 4.49

(tlj 2H, H-5 8 j b), 4.35(dt, 1H, J4>38H2, J4>58(b5Hz, H-4),

4.10 (dd, 1H| J3 24.5 Hz, J, 48Hz, H-3), 2.12 and 1.91(2s, 6H,

2 CH^CO-). inel.'calcd. for C16H16N409(4O8.3):47.06%C, 3.95%H,

13.72%N; Found: 46.90%C, 3.98%H, 157?N

3'-Acetamido-3'-deoxyadenoslne (III)

A'aplution of N -benzoyl-N , 9-bis(trimethylsilyl)edenine

(prepared of 3.65 g of N -benzoyladenine) in dry 1,2-dichloro—

ethaae (20 ml) was added to a solution of I (65 g) in a mix-

ture of dry 1,2-dichlor0ethane (60 ml) and dry ecetonitrile

(20 ml). A solution of stannic chloride (2 ml) in dry 1,2-di-

chloroethene was then added to the mixture. The reaction mix-

ture was heated at reflux for 8 hrs, cooled, diluted with

50 ml of chloroform, neutralized with saturated sodium hydro-

gen caibonate (100 ml) and filtered through a layer of Super-

cel Hyflo. The organic layer was separated, washed with satu-

rated sodium hydrogen carbonate, dried over Na2Su4 and evapo-

rated to dryness. The saturated at 0° solution of ammonia in

methanol (75 ml) was added to the residue and the mixture was

636



kept for 30 hrs at room temperature. The solution was evapo-

rated to diyness and the residue was washed with ether

(5 x 25 ml). The waaHings were combined, extracted with

50 ml of water. The aqueous phase was separated, combined

with the precipitate and evaporated to dryness. The residue

was crystallized from ethanol. Yield 2.2 g. The mother liquor

was evaporated, the residue was dissolved in water end appli-

ed to the Dowex-50 (H+) column ( 3 x 3 cm). The resin was

washed with a 30% ethanol (250 ml) and III was eluted with a

3% aqueous ammonia. The eluate was evaporated to dryness.

Crystallization of the residue from ethanol afforded additio-

nal 0.8 g of II. The total yield of III was 3 g (63.7%);

m.p. 24-7-249° dec (lit. 1 7 249° dec).

3'-Acetamldo-3'-deoxycytidine (IV)

A solution of N , 2-bis(trimethylsilyl)cytosine (prepa-

red of 0.99 g cytosine) in dry 1,2-dichloroethane (20 ml) was

added to a solution of I (4.26 g) in a mixture of di^ 1,2-di-

chloroethane (40 ml) and acetonitrile (20 ml). A solution of

stannic chloride (1.5 nil) in dry dichloroethane (10 ml) was

then added to- the mixture. The reaction mixture was heated

at reflux for 1.5 hr, cooled, diluted with 30 ml of chloro-

form, neutralized with saturated sodium hydrogen carbonate

(75 ml), filtered through a layer of Super-eel Hyflo and eva-

porated to dryness. A 10% aqueous ammonia (100 ml) was then

added to a residue and the mixture was kept overnight at room

temperature. The solution was evaporated to dryness, the resi-

due dissolved in 75 ml of water and applied to the Dowex-50

(H+) column (5 x 15 cm). The resin was washed with 0.5 1 of

water and the substance was eluted with a 3% ammonium hydro-

xide. The solution was evaporated to dryness, the residue

dissolved in the minimal amount of water and applied to the

Dowex 1 x 4 (0H~) column (4 x 10 cm) and eluted with water.

The fractions, containing the substance with Rp 0.52 in A

were collected and freese-dried. The yield of IV was 1.71 g

(60%).

637



3'-Azido-3'-deoxy8deno3ine (V)

To a solution of II (5.4 g) and N -benzoyl-N , 9-bis(tri-

methyl8ilyl)adenine (5.4 g) in dry 1,2-dichloroethane (100 ml)

a solution of stannic chloride (2 ml) in dry 1,2-dichloroetha-

ne (10 ml) was added and the mixture was heated at reflux for

2 hrs. The mixture was than cooled, diluted with chloroform

(100 ml), neutralized with saturated sodium hydrogen carbonate

solution (150 ml) and filtered through a layer of Super-eel

Hyflo. The organic layer was separated, dried over Na2S0^ and

evaporated to dryness. The saturated at 0 solution of ammonia

in methanol (75 nil) was then added to the residue, the mixture

was kept for 3 hrs Bt room temperature and evaporated to dry-

ness. The residue was washed with ether (3x30 ml). The wash-

ings were combined, extracted with water (100 ml). The aqueous

layer was separated, combined with the precipitate and evapo-

rated. Tê residue was dissolved in a minimal amount of hot

water and applied to the Dowex-50 (H+) column (3 x 10 cm). The

resin was thoroughly washed with a 30% aqueous ethanol and the

substance was eluted with the 23% NH^GH-ethanol-water (1:2:7).

The solvents were evaporated and the residue was crystallized

from water. The yield of V was 2.65 g (68%). LI.p. 219-220°

(lit 1 2 218-220°). Anal. Calcd. for C^H^GgN-j (292.2): 41.10%

C, 4.14%H, 38.34^; found: 41.OO%C, 4.17#H, 38.2'ijffli.

3'-Azido-3'-deoxycytidine (VI)

To a solution of N , 2-bis(trimethylsilyl)cytosine (pre-

pared of 0.6 g of cytosine) and II (2.1 g) in dry 1,2-dichlo-

roethene (50 ml) a solution of stannic chloride (1 ml) in

1,2-dichloroethane (8 ml) was 8dded and the mixture was heated

at reflux for 1 hr. The mixture was then cooled, diluted with

chloroform (50 ml) and neutralized with the saturated sodium

hydrogen carbonate solution (75 ml). The organic layer was

separated end the aqueous phase was extracted with chloroform

(2 x 30 ml). The combined extracts were dried over NagSO^ end

evaporated. The saturated at 0° solution of ammonia in methenol

(50 ml) was then added to the residue, the mixture was kept

at room temperature overnight and evaporated. The residue was



dissolved in the methanol-water (1:4) mixture, and applied to

the Dowex-50 (H+) column (2.5 x 4 cm). The resin wss washed

with water and the substance was eluted with a 3% solution of

ammonium hydroxide. The solution was evaporated to dryness,

dissolved in a minimal amount of ethanol and poured into ether

(50 ml). The residue was filtered off, dissolved in water and

freeze-dried. The yield of VI was 1.0 g (72.5%). Anal. Calcd.

for CglL^gO^. (268.23): 40.29%C, 4.47%H, 31-33%N; found:

40.18%C, 4.51%H, 3

3'-Azido-3'-deoxyuridine (IX)

To a solution of 2,4-bis(trimethylsilyl)uracil (prepared

of 1.35 g of uracil) and II (2.9 g) in dry acetonitrile (75

ml) a solution of stannic chloride (1.3 ml) in dry 1,2-dichlo-

roethene (2 ml) W8s added and the mixture was heated at reflux

for 1.5 h. The solvents were then evaporated, chloroform

(200 ml) and saturated solution of sodium hydrogen carbonate

(100 ml) were added to the residue and the mixture was filter-

ed through a layer of Super-eel Hyflo. The organic layer was

separated, the water layer was extracted with chloroform

(50 ml). The extracts were combined, dried over Na-SO^ and

evaporated to dryness. A saturated at 0° solution of ammonia

in methanol {7^ ml) was then added to the residue, the mixture

was kept at room temperature overnight, and evaporated to dry-

ness. The residue was dissolved in a mixture ethanol-water

(1:1) and applied to the Dowex 1 x 4 (0H~) column (2.5 x 7 cm).

The resin was washed with a 50% aqueous eth8nol and the sub-

stance was eluted with 0.1 11 solution of ammonium hydrogen

carbonate. The fractions, containing IX, were combined, evapo-

rated and the residue was several times co-evaporated with

water, dissolved in water (30 ml) and freeze-dried. The yield

of IX was 2.1 g (97%). Anal. Calcd. for C Q H ^ N C O C (269.22):

40.15SSC, 4.12%H, 26.02?SN; found: 4O.13%C, 4.16%H, 29.98591.

3'-Amino—3'-deoxyadenosine (VII)

Llethod A. A 2 U solution of sodium methylate in methanol

(60 ml) was added to III (3.0 g) and the mixture was heated at

639



reflux for 24 hrs. The mixture was cooled, neutralized with

Dowex-50 (H+) and was applied to the column, containing addi-

tional 10 ml of Dowex-50 (H +). The resin was washed with 100

ml of water and the product was eluted with 3% NHÔH in water.

The solution was evaporated to dryness and the residue was

washed with hot ethanol, then with ether and dried in vacuo.

The yield of VII was 1.1 g (43%), m.p. 260° (lit. 1 5 260°).

Method B. To a solution of V (2 g) in pyridine (60 ml)

triphenylphosphine (10 g) was added and the mixture "wss kept

for 3 hrs at room temperature. A 25% aqueous NILUH (40 ml)

was then added to the mixture, it was kept at room temperature

for 2 hrs and evaporated to dryness. The residue was washed

several times with hot ethanol, then with ether and dried in

vecuo. The yield of VII was 1.65 g (90.5%), m.p. 260° (lit.12

260°). Anal. Calcd. for C , , Q H ^ N 6 O , (266.26): 45.11%C, 5.30%H,

3L57%N; found: 44.93%C, 5.48%H, 31.31%N.

3'-Amino-3'-deoxycytidine (VIII)

Method A. A 2 M solution of sodium methylate in methanol

(30 ml) was added to IV (1.7 g) and the mixture was heated at

reflux for 24 hrs. The mixture was cooled, neutralized with

Dowex-50 (H+) end applied to the column, containing additional

5 ml of Dowex-50 (H +). The resin was washed with 100 ml of

water and the products were eluted with a y/o NHÔH. The solu-

tion was evaporated to dryness and the residue dissolved in

2 ml of water and applied to the silica gel column (2.5x20 cm),

equilibrated with the system ethenol-n-butanol Os'O* The

elution was run at first with 150 ml of the same system, the

3 ml fractions being collected. The coloured fractions were

removed, end the colourless, containing IV combined. The fur-

ther elution (using 150 ml of ethanol) gave the residual IV.

All the fractions containing IV were combined and evaporated

to dryness. The recovery of IV was 0.31 g. The compound VIII

was eluted with 250 ml of a 85% aqueous ethanol. The solution

was evaporated to dryness. The residue was dissolved in water

(250 ml) end freese-dried. The yield of VIII.was O.56 g (39%).

Llethod B. To a solution of VI (268 mg) in pyridine (4 ml)

triphenylphosphine (1.5 g) was added end the mixture was kept

640



at room temperature for 3 hrs. A 25% aqueous NiLOH (2 ml) was

then added to the mixture, which was kept at room temperature

for additional 2 hrs end finally evaporated to dryness. b'ther

(50 ml) and water (50 ml) were then added to the residue, the

aqueous layer was separated and eyaporeted to dryness. The

residue was dissolved in minimal amount of ethanol and the

solution was added dropwise into 80 ml of ether. The residue

was filtered off, dissolved in water (25 ml) and freeze-dried.

The yield of VIII was 184 mg (76%). Anal Calcd. for CglL^NÔ^

(242.23): 44.62%C, 5.83%H, 23.13%N; found: 44.59%C, 5.86%H,

23.21JBT.

3'-Amino—3'-deoxyuridine (X)

To a solution of IX (54 mg) in a mixture of pyridine -

25% aqueous ammonium hydroxide (1:1,2 ml) triphenylphosphine

(I56 mg) was added, the mixture was kept for 2 hrs at room

temperature, end evaporated to dryness. Water (20 ml) and

ether (20 ml) were then added to the residue, the water layer

W8S separated, washed with ether (3 x 15 ml), concentrated

to a 10 ml volume and freeze-dried. The yield of X was 46 mg

(94%). Anal. Calcd. for C g l L , ^ ^ (243.22): 44.44%C, 5.39%H,

17.28%N; found: 34.40%C, 5.42%H, 17.21N.

3'-Azido-3'-deoxynucleoside 5'-phosphates (XXII-XXIV)

A cooled to G° solution of POC1~ (0.4 ml) in triethy1-

phosphate (20 ml) was 8dded to a nucleoside (2 mmole). The

mixture was kept for 24 hrs at 4° and finally neutralized with

25% aqueous ammonium hydroxide and left for additional 1 h at

4°. Water (50 ml) was then added, and the mixture was extracted

with benzene (25 ml) and then with ether (2 x 25 ml). The

water layer was separated, evaporated to dryness and dissolved

in water (200 ml). The further isolation of nucleotides was

run in the following way. The solutions of XXII and XXIII in

water were applied to Dowex-50 (H+) columns (2.5 x 10 cm).

The elution was performed using water. The UV-absorbing frac-

tions were collected, combined, evaporated to dryness, co-eva-

porated with a 10% aqueous ammonium hydroxide (15 ml) dissolv-

641



ed in wster (25 ml) and freeze-dried. The yield of XXII was

73%. The yield of XXIII was 58%.

The solution of XXIV in water was applied to the DE-32,

column (5 x 25 cm). The elution W8S performed with a linear

gradient of concentration (0.0 M —• 0.2 M, V = 7.5 1) of ammo-

nium hydrogen carbonate buffer. The fractions containing XXIV

(the 0.11 II concentration of ammonium hydrogen carbonate) were

combined, evaporated to dryness, several times co-evaporsted

with water and freeze-dried. The yield of XXIV was 39%.

3'-Amino-3'-deoxynucleo3ide 5'-phosphate3 (XXV-XXVII)

To a solution of ezidonucleotide (XXII-XXIV, 1 mmole) in

the mixture of pyridine (15 ml) and a 25% aqueous ammonium

hydroxide (10 ml) triphenjlphosphine (800 mg) was added and

the mixture was kept overnight at room temperature. The sol-

vents were then evaporated to dryneas, water (50 ml) and

ether (50 ml) were added to the residue. The water layer was

separated, washed with ether (2 x 25 ml), concentrated to a

20 ml volume and freeze-dried. Yields: 97>a of XXV, 83% of

XXVI, S4f0 of XXVII.

ACKUOULbDGJiL&NT

The authors are thankful to Dr. V.L.Florentiev for his

constant interest and to Mrs. G.V.Iioussinova for preparation

of the manuscript.

1 For part III see: Azha,/ev, A.V., Kreyevsky, A.A., Smrt, J.(1978) Coll. Czech. Chem. Cormun. 4J, 1647-1654

2 Freser, Th. h., M c h , A. (1975) Proc. Hat. iced. Sci. USA72, 3044-3047

3 Armstrong, V..(., Isckstein, F. (1976) ±iUr. J. Biochem. '70,33-38

4 Nathans, D. (1967) in Antibiotics, Lechanism of Action,Gotlieb, D., Shaw, P., ids, Vol. 1, 251-267

5 Tê results v;ill be published elsewhere6 Popovkina, S.V., Azhayev, A.V., Kharshan, U.A., Krayevsky,

A.A., Bobruskin, I.D., Gottish, E.P. (1979) Bioorg. Chem.(Russian), in Press

7 Azhayev, A.V., Smrt, J. (1978) Coll. Czech. Chem. Comnun.4J, 1520-1530

642



8 hi Khadem, H.S., Audichya, T.D.. £1 Ashry, L.S.H., Sindric,R. (1975) Carbohydr. Res. 41, 318-322

9 Lichtentheler. F.W., Voss,~F., Heerd, A. (1974) TetrahedronLett. 74, 2141-2144 and other literature cited there

10 NiedbalTa, U. , Vorbriiggen, H. (1976) J. Org. Chem. 41,2084-2092

11 Tazewa, J., Tezawe, S., Alderfer, J.L., Ts'o, P.O.P.(1972)Biochemistry 11, 4931-4939

12 Mengel, R., WTedner, H. (1976) Chem. Ber. 10^, 433-44313 Altona, C , Sundaralingam, M. (1973) J- -Amer. Chem. Soc.

55, 2333-234414 Uerna, J. (1975) F2BS Lett. 58, 94-9815 Kotusov, V.V., Kukhanove, M.K7, Victorova, L.S., Krayevsky,

A.A., Treboganov, A.D., Gnuchev, N.V. , Florentiev, V.L.,Gottikh, B.P. (1976) Molecular Biology (Russian) 10, 1394-1401 —

16 Baker, B.R., Shaub, H.m. (1955) J. Amer. Chem. Soc. 72,5900-5905 ~

17 Baker, B.R., Sheub, R.E., Kissman, H.M. (1955) J. Amer.Chem. Soc. 71, 5911-5915

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Documents

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