scale column chromatography is, however, timc conand degradasuming and can lead to tions; consequently, a new method was developed. Purification of isomerically pure (i.e., one internucleotide linkage type) products was carried out cont'inuously on the Hannig cont,inuous-flow elect'rophoresis (cfe) apparat,us which employs no stationary phase.4 All nnprot ected phosphorus-ront airiirig proclurt's were c$haT:tcterized by their hydrolytic. stability toimrd the mono- and diesterase of rattlesnake ~ e n o i i i ,hoviiie spleen diesterase, ribonuclease I, xnd b:tcteriw.l alkaline hatase. Sim spectroscopy w:ts eniployed to tmhestructures of dl of the phosphorus-c.oiitxiriing product,s. Three routes were employed for dinucleoside phosphate synthesis. The first' yielded a mixture of 2'+5' and 3'+5' isomers (both isomers were desired for biochemical and biological evaluation). A nucleoside (I) with the 2' (and 3') hydroxyls unprotect,ed was vondensed viit,h a fully protected ,5'-nucleotidr 11. The proterting groups were then removed from the intermediate without prior purihcat'ion by wnsecutiue base and arid hydrolysis to give the desired procliwts 111 and 11: (see route 1) whicbh were t,lieii sep:ii~tedhy :L suit'able procedure (see Table I). TOCH,
A'
HO )i
w
HOCHz A'
RO Y '1
protected f
I1
-+
dinucleoside phosphate
CHZOP(0)OH -+
I
w
CHzO B
(2)
HO X VI
The third sequence afforded only symmetrical 5'+.5'dinucleoside phosphates and phenyl 5'-phosph:ites. The 5'-unprotected iiucleoside (T-) w a s coiidensed with phenyl phosphodivhlorodate according to A\Iichelson"ato give products of type T'II and T'III after alkaline hydrolysis. HO X
H
(HO)aP(0)0c Hz B '
w+ P
condensation
HOI X
HI1 Y
protected dinucleoside phosphates
How +
Q
HO Y
111, 3'-+5'
1'11
hydrolysis
w
HOCHz
A
HOI b P ( 0 j O H CHzO B
(1)
HO Y ' IV, 2'-+5'
T = trityl
01' moiiomethos?-tiityl; B' = hriitable proiected piiriiie or pyrimidiiie baheh; X = I-' = a-OH, ,!%OH, 01'H ; Y = aryl POH, aryl p-OH, o r H ; 11 = acayl
Route 2 produced only 5'+5'-linlied symmetriral or unsymmetrical diriucaleoside phosphates. Coridensat,ion of 5'-unprotected nucleosides (1') with protected nucleotides as I1 above followed by hase hydrolysis gave an isomerically pure product T'I vhirh could then he purified by cfe. (3) For leading references before 1962 see (a) H . G . Khurana, "Some Recent Ilevelopments in the Chemistry oi Phosphate Esters of I4iological Interest," John IYiIej- and Sons, Inc., New York, X. T.. 1961, Ctiagters 2, 5 , and 6 : (b) -1,11. 3Iiclielson, "The Chemistry of Nucleosides and Nucleotides," Academic Press Inc., Xew York, N . Y.,1963. More recent v o r k is found in (cj SI. Smith, D. H. Rammler, I. H. Goldberg. and H . G . Khorana, J . A m . Chem. SOC., 84, 480 (1962); (d) I). H. Rammler a n d H. G. Iiliorana, ibid., 3112 (19621; (e) G. \Yeimann and H. G. Khoranrt. i b ~ d . ,1329 11962); ( f ) 11. H.Rammler, I-.Lapidot, and H. G . Khorana, i h i d . , 86, 1989 (1963); ( n ) H. Bchaller, G . \Veimann, H . Lach, and H. G . Iihorana, ihid., 86, 3821 (1963); (11) H. Schaller and H. G . Khorana, i h i d . , 8 5 , 8828 (196:3); (ii 'i. Lapidot and H. G. Khorana, i h i d . , 86, 3852 (1963); ( j j R. Lohrmann and H. G. Khorana, ibid., 86, 4188 (1964). (4) Details of this method will be published separately h y H. KO, 11. K q w , and \V. J. Il-pchter, B n n l . Riochrm., in press.
The required protected nucleosides arid nucleotide derivative;< of am-cytidine were prepared as outlined in Chart, I.fi am-Cytidine (1) was nionotrit,ylated in good yield giving the ether TrCA (2). Renzoylation followed by acid hydrolysis wit'hout isolation of the int'erniediate gave the t8ribenzoate (3). a type T' intermediate. Phosphorylation according to Tener"b followed by t'he usual base hydrolysis afforded the crystalline S4-benzoyl 5'-phosphate 4. dcetylation of' this product gave t'he desired prot'ected nucleotide 5 . a type I1 int'ermediate. Alternatively, anisoylation of CX followed by base hydrolysis afforded S4-anisoy1-aracyt'idine (6) in relatively poor yield. Tritylation of this subst'ance with p-anisyldiphenylniethyl chloride gave the methoxytrityl ether 7 (a type I intermediate) which could not be crystallized. but was homogeneous by tlc. This subst'arice was converted t.0 a type T7 iritmerniediat'eby conserutive henzoylation and acid hydrolysis t'o give t'he dibenzoyl derivat'ive (8). again in poor yield. Consequently, the more desirable type I int'ermediate was obtained by the benzoylation of TrCA (2) followed by hydrolysis giving the crystalline S4-benzoyl-5'-O-trityl-a~a-cytidine(9). We then had the necessary protected derivatives of am-cytidine for condensation with known protected derivatives of the natural nucleosides and nwleotides to ( w r y out nll dinurleosi de phosphate syntheses. (.i) hi .\. 11. 3Iiclielwn, RioclrirrL. Biopiius. .lct,i. 66. 841 i l U 6 2 ) ; ( i l l G . 11. Tener. . I . . I m . C h e m . ~ ' O C . , 8 3 , 159 (1Y61). (6) The sliorthand alihreviationa used in this payer are a6 iolloHs: tlic 5' linkage is t o the iefr of CI,and tlie 3' linkage folloxed iiy the ?' linkagc and tile hase sulmtitrient is to the right of C.i. .\lea, a p t o the left of the symbol (C.1, etc) indicates a 5' linkage, a superscript p t o the right of t h r symbol indicates a 2' linkage, and a siihscript p t o the rinht of rhe a.ml,ol indicates a 3' linkage.
K L‘CLKIC 1 ~ C I l ~ S .1
_______ I
x
Tlc, H P or re1 K f
10-2
c 0.22
c 0.21 c 1 , o re1 tu c,c See no. 12 c 0.S6 c0.sti S 1.30re1 to c,u Cf 0.3ti C 0.41 C 0.39 C 0.98 2)s. C,A C 1.1 us. A,C C 1 . 1 us. A & C I . 36 us. C,4 c 1.2.5 us. C,A cu 1.1 U S . u,c CQ1.03cs. U,C
-18 -1s
-22
70”
1.8 14. 10.0 3,;. 0 1.28 :3. 83 s3.0 9.3 8.7 23.0
3Y.U
11 . o !) . 9
CO.Yus.C,U
10.5
S 1.27re1 to U , C S 1 . 2 7 re1 t o U,C CO.33
4 14 20 42
S 1 , O re1 to C,C C 0.8vel to pCXCh
50
co.24 C1.0reitoCA S0.54
live rrl t o stdd
Starting illateriais”------------
Coinpd
Compd
110.
no.
l’rotec t ed nucleoside
J
9
TrCA(SBz)
> )
7
CA(BzBzSBz) .\ltCA(SRhj
7
.\ It CA (KAII )
J .22 b See no. 12 1 , 07 c
.-
4 bd
1 . 0 ~ U.97 c
,
1.1 c 1.24d) l.OSd, 0.97e
0
C.l(BzHzSBsj CA(BzBzSI3/.)
9
TICA(NBL)
>
C A (BzH6S B z )
1 .i 7.0 4 . .i
c1.ocs.c,u
26.6
12.6
Yield.
?.-
3
