Stereoconfiguration markedly affects the biochemical and biological properties of phosphorothioate analogs of 2-5A core, (A2'p5')2A.

The diester bonds of phosphorothioate trimer analogs of (A2'p5')2A (2-5A core) of the Sp stereoconfiguration were found to be extremely stable to hydrolysis by both serum and cellular phosphodiesterases. The corresponding Rp isomers, although still more stable than parent ppp(A2'p5')2A (2-5A), were significantly more susceptible to enzymatic hydrolysis than were the Sp isomers. Utilization of these novel 2-5A trimer isomers containing various combinations of Sp or Rp configurations at the internucleotidic phosphorothioate linkages revealed a further specificity of this enzymatic hydrolysis. Thus, the stereoconfiguration of the bond adjacent to the one undergoing hydrolysis influenced the rate of enzymatic hydrolysis, as well as did the chain length of the oligomer. The most stable trimer analog, which contained both internucleotide phosphorothioate linkages of the Sp configuration, had a half-life of 30 days in serum, which is a 1500-fold increase over that of parent 2-5A core. This is the first report on biochemical stability of an oligonucleotide containing more than one phosphorothioate linkage of the Sp configuration and is the first demonstration that a phosphorothioate internucleotide bond of the Sp configuration can increase the enzymatic stability of an adjacent phosphorothioate bond. In marked contrast to all previous 2-5A core analogs of increased stability, the activity (antiproliferative and antiviral) of the stable phosphorothioate 2-5A core analogs was obtained with the intact trimer, i.e., it was not attributed to antimetabolite degradation products.

The diester bonds of phosphorothioate trimer analogs of (AZ'p5')%A (2-5A core) of the S, stereoconfiguration were found to be extremely stable to hydrolysis by both serum and cellular phosphodiesterases.The corresponding R, isomers, although still more stable than parent p~p(AZ'p5')~A (2-5A), were significantly more susceptible to enzymatic hydrolysis than were the S, isomers. Utilization of these novel 2-5A trimer isomers containing various combinations of S, or R, configurations at the internucleotidic phosphorothioate linkages revealed a further specificity of this enzymatic hydrolysis. Thus, the stereocontiguration of the bond adjacent to the one undergoing hydrolysis influenced the rate of enzymatic hydrolysis, as well as did the chain length of the oligomer. The most stable trimer analog, which contained both internucleotide phosphorothioate linkages of the S, configuration, had a half-life of 30 days in serum, which is a 1500-fold increase over that of parent 2-5A core. This is the first report on biochemical stability of an oligonucleotide containing more than one phosphorothioate linkage of the S, configuration and is the first demonstration that a phosphorothioate internucleotide bond of the S, configuration can increase the enzymatic stability of an adjacent phosphorothioate bond. In marked contrast to all previous 2-5A core analogs of increased stability, the activity (antiproliferative and antiviral) of the stable phosphorothioate 2-5A core analogs was obtained with the intact trimer, i.e., it was not attributed to antimetabolite degradation products.
Interferon (a, p, or y) treatment of many cells in culture results in a multitude of biochemical changes, including the induction of several proteins, and ultimately results in the establishment of an antiviral state, inhibition of cell proliferation, and, in certain relevant cells, immunomodulatory effects (reviewed in Stewart, 1981). One of the proteins induced after interferon treatment is an enzyme, 2-5A1 synthetase, which is activated upon binding to double-stranded RNA (Baglioni et al., 1981b;Torrence et al., 1981). The activated enzyme polymerizes ATP into a series of 2'-5'-linked adenylate oligonucleotides containing a triphosphate at the 5' terminus (Ball and White, 1978;Kerr and Brown, 1978;and reviewed in Baglioni, 1979;Lengyel, 1982;. Oligomers containing a 5'-dior triphosphate and three * This is Contribution 213 from the Institute of Bio-Organic Chemistry. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'The abbreviations used are: 2-5A, ppp(A2'~5')~A, 2-5A core, (A2'~5')zA. or more monomer units bind to, and subsequently reversibly activate, an endogenous or sometimes interferon-induced (Jacobsen et al., 1983aSilverman et al., 1983a) endoribonuclease (Baglioni et al., 1978;Ball and White, 1979;Clemens and Williams, 1978;Eppstein et al., 1979;Eppstein and Samuel, 1978;Ratner et al., 1978;Williams et al., 1979a). The activated endonuclease cleaves messenger and ribosomal RNAs Wreschner et al., 1981), resulting in inhibition of translation. The 2-5A molecules are readily cleaved by cellular phosphodiesterases, and thus their ability to inhibit protein synthesis is transitory . The 2-5A system has been implicated as contributing to the antiviral state in several cell-virus systems (reviewed in Baglioni, 1979;Lebleu and Content, 1982;Lengyel, 1981Lengyel, , 1982Revel, 1979;Torrence, 1982). The 2-5A core, which lacks the terminal 5'-phosphates, does not bind to (Knight et al., 1980) or activate the endonuclease (Williams and Kerr, 1978;Eppstein et al., 198313). Recently, the detection of high concentrations of 2-5A core in intact cells (Kerr et al., 1983;Knight et al., 1980) raises the question of its biological significance. However, meaningful studies have been hampered by its extremely short half-life, and thus a stabilized analog is desirable.
In order to study the 2-5A system, we and others have employed analogs of 2-5A containing modifications primarily on the sugar moiety (Baglioni et al., 1981a;Chapekar and Glazer, 1983;Doetsch et al., 1981;Eppstein et al., l982,1983aEppstein et al., l982, , 1985Imai et al., 1982;Lee and Suhadolnik, 1983;Sawai et al., 1983;Suhadolnik et al., 1983), on the base (Drocourt et ul., 1982;Torrence et al., 1984)) or changing the 2'-5' phosphodiester linkage to a 3'-5' linkage (Lesiak et al., 1983). Initial studies suggested that analogs of 2-5A core, which contained modified sugar residues and were stabilized to degradation by cellular phosphodiesterases, showed significant antiproliferative and antiviral activities on nonpermeabilized cells, in contrast to the unstable parent 2-5A core. However, it was subsequently demonstrated that all the biological activities of these "stable" 2-5A core analogs were attributable to the antimetabolite degradation products produced as a consequence of hydrolysis by serum enzymes (Chapekar and Glazer, 1983;Eppstein et al., 1983a). We wished to study an analog of 2-5A core that was stabilized both to cellular as well as serum phosphodiesterases and which, even if it were eventually degraded, would not yield antimetabolite nucleosides. Such an analog could provide a handle in studying, in the intact cell system, whether intrinsic biological activities of 2-5A core existed which were unrelated to endonuclease activation. We chose to study phosphorothioate derivatives of 2-5A core as candidates for meeting the above requisites.
It has been determined for some polynucleotide hydrolases that phosphorothioate internucleotide linkages of either the 5999 R, or S, configuration are stabilized to varying degrees against enzymatic hydrolysis (Eckstein, 1985). We wished to determine what the effects of two adjacent phosphorothioate internucleotide linkages (containing combinations of R, and s, configurations) were on enzymatic hydrolysis of 2-5A core analogs. Due to the acceptance by polynucleotide polymerases of phosphorothioate nucleotides of the S, but not R, stereoconfiguration, coupled with the inversion of configuration that occurs during the enzymatic polymerization, oligonucleotides containing phosphorothioate internucleotide linkages of the S, configuration have not been obtainable by enzymatic synthesis (Eckstein, 1985). For example, Lee and Suhadolnik (1985) recently reported on the enzymatic synthesis of phosphorothioate 2-5A (from a-thio[S,]ATP) which contained internucleotidic phosphorothioate bonds of the inverted R, configuration.
We now have utilized novel chemically synthesized 2-5A analogs which are modified at the 2'-5' phosphodiester linkage only, by the substitution of a sulfur for an oxygen (Nelson et al., 1984). This substituted phosphorous is chiral and exists in either R, or S, stereoconfiguration. Consequently, the resulting core analogs (i.e., lacking the 5"triphosphate) of trimer 2-5A contain two such chiral centers and can exist as four stereoisomers (R$,, SJ?,, S,S,, R,S,). We have utilized this novel set of analogs to analyze the specificity of the cellular and serum phosphodiesterases that degrade the 2-5A species via hydrolysis of the phosphodiester bonds. We report that, in addition to a stereoconfiguration preference of the phosphodiester bond undergoing hydrolysis, the phosphodiesterases also recognize the configuration of the phosphodiester bond adjacent to the bond undergoing hydrolysis. The result is that the analog most stable to cleavage contains the S, configuration at both phosphodiester bonds. The biological activity of these novel stable 2-5A core analogs on intact cells is also determined. For the first time, both antiproliferative and antiviral activities were obtained which were attributable to the intact stabilized 2-5A trimer core.

DISCUSSION
Previous studies employing polynucleotides containing the phosphorothioate diester linkage have been limited to enzymatically synthesized polymers with the R, stereoconfiguration. To date all polymerases studied only accept the nucleoside 5'-O-(l-thiophosphates) of the S, stereoconfiguration and result in inversion of configuration at the phosphorus in their polymerization (Eckstein, 1985;Lee and Suhadolnik, 1985). Our present studies utilizing chemically synthesized trinucleotides allow us to examine for the first time the influence of adjacent S, versus R, stereoconfigurations on the enzymatic stability at the phosphorothioate bond.
We now show that in addition, the stereoconfiguration of a modified phosphodiester linkage significantly affects its enzymatic stability, as also does the substrate chain length. Thus, maximally productive interaction of both cellular and serum phosphodiesterases with 2-5A molecules has the appearance of involving multiple "subsites" on the enzyme such as described for other hydrolases (Allen, 1980). The stereoconfiguration of the phosphorothioate linkage increased the stability to all phosphodiesterases tested, with the S, conformation significantly more stable than the R, (Table 1, Fig. 2).
Most interestingly, this same effect was also observed with the phosphorothioate linkage adjacent to the one undergoing hydrolysis. That is, the trimer S,S, was more stable than the trimer R,S, (Table 1, Fig. 2), even though in both cases the bond undergoing hydrolysis was of identical S, configuration.
The cellular phosphodiesterases showed more discrimination as to substrate specificity than did the serum enzymes, as illustrated by the greater than 360-fold increased stability of the phosphorothioate dimer R, isomer over the parent 2-5A trimer in cell extracts versus only a 5-fold stability differential for these same molecules in serum ( Table 1 and Fig. 4). Such results are in parallel with our previous findings of the reduced stability of xyloadenosine 2-5A analogs to serum versus cellular enzymes (Eppstein et al., 1983a).
Previous studies employing 2-5A core analogs of increased metabolic stability have shown marked antiviral and antiproliferative activities. However, upon analysis, it was confirmed that all these activities were due to the antimetabolite effects of nucleoside analogs obtained as degradation products, both for the cordycepin 2-5A cores (Chapekar and Glazer, 1983) and the xyloadenosine 2-5A core analogs (Eppstein et al., 1983a). Accordingly, the antimetabolite activities were markedly increased in the presence of the adenosine deaminase inhibitors coformycin or deoxycoformycin. The availability now for the first time of 2-5A trimer core analogs that are extremely stable to both serum as well as cellular phosphodiesterases and which would not yield antimetabolite nucleosides even if they were to be degraded allowed us to assess what biological activity (if any) could be attributed to an intact 2-5A core species after treatment of nonpermeabilized cells. Anticellular and antiviral activities were obtained, but only with 25-50 PM phosphorothioate 2-5A core analogs. The more stable S, isomers were more active than the R, isomers.
As predicted, ribosomal RNA analysis of cells treated either with the phosphorothioate analogs of 2-5A or with parent 2-5A core (Eppstein et al., 1983b) did not show significant degradation of the rRNA such as is obtained when parent 2-5A 5'-triphosphate is introduced into cells via calcium phosphate coprecipitation. Thus, the activity obtained with the 2-5A core analogs does not appear to be due to activation of the 2-5A-dependent endonuclease.
We can for the first time conclude that antiviral and antiproliferative activities are obtained with intact 2-5A core species on nonpermeabilized cells. In contrast to previous studies, these activities are not mediated through various degradation products. The activity of 2-5A cores, which do not activate the endonuclease in these nonpermeabilized cells, is much lower than the activity of 5"triphosphate 2-5A in cell-free extracts or after calcium-phosphate precipitation onto intact cells. However, it is not known how much (if any) of these externally added 2-5A core analogs actually penetrate into the cell. Our present results do not rule out an effect of 2-5A cores at the cell surface, for example by interacting with adenosine or ADP receptors. In another system, however, we have determined that phosphorothioate 2-5A cores do not inhibit ADP-induced platelet aggregation, such as is observed with adenosine or AMP.3 Lee and Suhadolnik (1985) recently reported that enzymatically synthesized 5'-triphosphate phosphorothioate 2-5A trimer and tetramer analogs, which contained the Rp configuration at the 2'4' phosphorothioate bonds, were more potent than parent 2-5A 5'-triphosphate in inhibiting protein synthesis in cell extracts. The 5"triphosphate of the more stable S, isomers is not yet available, as it cannot be prepared enzymatically and the chemical synthesis is not trivial. When it is available, future studies will determine what activities can be attributed to the 5'4riphosphates of the much more stable 2 ' 4 ' phosphorothioate S, isomers.  M). c a l f serum was frm K.C. Biological increased elution time of species containing the S configuration (panels A and 6 ) . The formation of inosine due to the action of phosphatEses. 5"nucleotidase and adenosine deaminase present i n t h e c e l l e x t r a c t o r serum was also observed when AMP was incubated i n these e n z p sources. The unlabeled peak i n panel 0 was not identifiable with our known standards. h a l f l i v e s were calculated from the degradation pattern of the trimer Sp mixture as shnm i n Figure 2. . and a f t e r 2 days incubation the activity was only half as potent as i n fresh extracts (Fig.   3 ) . S t a b i l i t y determinations i n c e l l e x t r a c t s were accordingly limited to 24 h r incubations. and thus the half-life calculations for the yew stable analogs are inherently less accurate.
However. the phosphodiesterases present i n the serum did not appear t o l o s e a c t i v i t y a f t e r f o u r days incubation a t 37-C (not sham).
Thus h a l f -l i f e determinations i n serum were based On a four dav incubation. and are accordingly more accurate for the very stable analogs. The phoSphorothioate dimer 2-5A analogs were found t o be considerably Rore stable than Were the corresponding trimer analogs (Table 1). This also was true far the parent 2-5A core oligomers h e n analyzed with cell extracts (Fig.  4). I n t h e presence of serum enzynes.
approximately twice that of the tetramer species. Overall. the parent 2-5A analogs were however. the parent 2-SA dimer and t r i m e r a n a l q s were o f comparable stability. vhich was analogs were mare stable i n c e l l e x t r a c t s than i n serum.
10-24 times more stable i n serum than i n cell-free extracts; whereas the phosphorothioate ., diner;

INFLUENCE OF SUBSTRATE CHAIN LENGTH ON
Bioloqical Activities. The effect of the various 2 -M trimer analogs on v i r a l and c e l l u l a r growth is sham i n Table 2. The trimer S mixture was comparable i n apparent potency t o parent 2-511 core f o r i n h i b i t i o n o f c e l l g!wth.
but it exerted a t r u e a n t i p r o l i f e r a t i v e adenosine deaminase, had no e f f e c t on t h e a c t i v i t y of the trimer Sp mixture (not shown).
effect rather than reducing c e l l v i a b i l i t y as d i d parent core. Coformycin. an i n h i b i t o r of The t r i m e r S mixture shoved greater antiviral activity against HSV-1 than did the trimer R mixture a! 50 VU. whire parent 2-511 core showed no a c t i v i t y (Table 2). No ribosomal REA degradation was detected a f t e r treatment Of cells with these 2-51 core analogs ( n o t shown).  h t e r f e r o n was nIH reference standard of mouse fibroblast interferon (-+ 8). XGWZ-904-511.