text:
Oligonucleotides possessing at least one sulfamate or sulfamide internucleotides linkages. These compounds can be used as specific hybridization probes to detect complementary nucleic acid sequences.
1. A compound of the formula: ##STR14## wherein: A is H, OH, OR8, OQ or halogen;
B is a naturally occurring nucleic acid base or modified nucleic acid base;
Y is --N(R)-- or O;
Z is --N(R7)-- or O;
wherein Y, OR2, and A are independently bonded to the 2' or 3' position and provided further that A is bonded to a position different from that of Y or OR2 ;
wherein each M is independently selected from ##STR15## provided at least one M is S(═O)═O; and provided that when M is S(═O)═O, Y and Z cannot both be O;
n is 1 to 75;
R is H or alkyl of 1 to 6 carbon atoms;
R1 is H2 PO3, H3 P2 O5, H4 P3 O7 and their suitable salts, H or an hydroxyl protecting group;
R2 is H2 PO3, H3 P2 O5, H4 P3 O7 and their suitable salts, H or an hydroxyl protecting group;
R3 is alkyl or cyanoethyl;
R4 is H or alkyl of 1 to 6 carbon atoms;
R5 is H or alkyl of 1 to 6 carbon atoms;
R6 is H or alkyl of 1 to 6 carbon atoms;
R7 is H or alkyl of 1 to 6 carbon atoms;
R8 is alkyl of 1 to 6 carbon atoms;
R9 is alkyl of 1 to 6 carbon atoms or cyanoethyl; and
Q is an hydroxyl protecting group;
provided that when each M is S(═O)═O, then at least one B group is not thymine.
2. The compound of claim 1 wherein when M is S(═O)═O, one of Y or Z is --N(R)-- or --N(R7)-- and R or R7 is H.
3. The compound of claim 1 wherein R is an alkyl of less than 5 carbon atoms.
4. The compound of claim 1 wherein R3 is an alkyl of less than 5 carbon atoms.
5. The compound of claim 1 wherein R4 is an alkyl of less than 5 carbon atoms.
6. The compound of claim 1 wherein R5 is an alkyl of less than 5 carbon atoms.
7. The compound of claim 1 wherein R6 is an alkyl of less than 5 carbon atoms.
8. The compound of claim 1 wherein R7 is an alkyl of less than 5 carbon atoms.
9. The compound of claim 1 wherein R8 is an alkyl of less than 5 carbon atoms.
10. The compound of claim 1 wherein R9 is an alkyl of less than 5 carbon atoms.
11. The compound of claim 1 wherein the A substituent is in the 2'-position.
12. The compound of claim 1 wherein A is selected from the group consisting of H and OH.
13. The compound of claim 1 wherein B is adenine, guanine, cytosine, thymine or uracil.
14. The compound of claim 1 wherein M is S(═O)═O for the internucleotide linkage at the 3' terminus or 5' terminus, or both the 3' terminus and the 5' terminus of the compound.
15. The compound of claim 1 wherein M is S(═O)═O.
16. The compound of claim 1 wherein M is S(═O)═O or P(═O)--OH.
17. The compound of claim 16 wherein Y or Z is --N(R)-- or --N(R7)--, and R or R7 is H.
18. A compound of the formula: ##STR16## wherein: A is H, OH, OR4, OQ, or halogen;
B is a naturally occurring nucleic acid base or modified nucleic acid base;
n is 1 to 75;
R1 is alkyl of 1 to 6 carbon atoms;
R2 is dialkylamino, morpholino, piperidino, or pyrrolidono;
R3 is alkyl of 1 to 6 carbon atoms;
R4 is alkyl of 1 to 6 carbon atoms;
Y is --N(R)-- or O;
Z is --N(R)-- or O;
wherein Y, OP(R2)(OV), and A are independently bonded to the 2' or 3' position and provided further that A is bonded to a position different from that of Y or OP(R2) (OV);
R is H or alkyl of 1 to 6 carbon atoms;
Q is an hydroxyl protecting group; and
V is an hydroxyl protecting group;
provided that at least one B group is not thymine.
19. The compound of claim 18 wherein V is cyanoethyl or methyl.
20. The compound of claim 18 wherein Q is selected from the group consisting of C1 -C5 trialkylsilyl, C1 -C5 alkyl, acyl, cyanoethyl or tetrahydropyran.
21. The compound of claim 18 wherein R is an alkyl of less than 5 carbon atoms.
22. The compound of claim 18 wherein R1 is an alkyl of less than 5 carbon atoms.
23. The compound of claim 18 wherein R3 is an alkyl of less than 5 carbon atoms.
24. The compound of claim 18 wherein R4 is an alkyl or less than 5 carbon atoms.
This invention relates to novel compounds in which one or more of the internucleotide phosphodiester linkages in oligonucleotide analogs have been replaced by a sulfur based linkage.
The repertoire of substances available for therapeutic purposes consists primarily of relatively low-molecular weight organic compounds. Recently, the repertoire has been expanded to include proteinaceous materials which have been engineered for efficacy, specificity and stability. Increasing attention is now being focused on the therapeutic potential of other classes of biomacromolecules, including nucleic acids.
Nucleic acids are linear phosphopentose polymers bearing pendant adenine (A), guanine (G), cytosine (C), and thymine (T) [or the related uracil (U)] base groups. The pentose may be ribose (RNA) or 2'deoxyribose (DNA). They are attractive candidates for therapeutics due to the high potential for selectivity. The basis for this high selectivity is the well-known ability of a nucleic acid to form an antiparallel, two-stranded, helical structure (or duplex) with its structural complement through the formation of hydrogen bonds between the bases on opposite strands (Watson-Crick base pairs). Complementarity is defined as the pairing of G with C and A with T [or U] on opposite strands. Duplexes with perfect complementarity are thermodynamically preferred. For short [<20 residues or nucleotides (nt)] oligonucleotides, a single improper pairing or mismatch can significantly destabilize the duplex. Thus one can, in principle, selectively address a single site in a 3�109 nt human genome (the genetic material of a human in its entirety) with an oligonucleotide of 16-20 nt. This is substantially greater selectivity than one can generally achieve with traditional, low-molecular weight agents. With this degree of potential selectivity, one can consider the approach of exerting a therapeutic effect at the level of gene expression. For viral agents which act through integration of their genetic material into the host system, one can envision blocking one of the many steps involved in integration and replication.
Most of the attempts to use nucleic acids as complementary addressed therapeutic agents have involved single-stranded targets. Such targets include messenger RNA (mRNA) and single-stranded viral genomes. In such cases, the reagent nucleic acids are complementary to the target and are referred to as "anti-sense reagents". The process of using such agents to exert a specific effect is referred to herein as "anti-sense targeting"1-3,32.
More recently, a second, high-specificity mode of nucleic acid binding has been investigated. It has been found that certain sequences of duplex DNA will bind a third strand to form a triple helix or triplex4. Triplex formation involves the formation of base triples with the additional base forming hydrogen bonds in the so-called Hoogsteen mode. Reagents designed to bind in such a mode are referred to herein as "triplex reagents" and the process of using such reagents to exert a specific effect will be referred to as "triplex targeting". The advantage of the triplex targeting approach is that one can address double-stranded genomic DNA directly. The disadvantage is that, at least at this time, not all sequences can be addressed in this fashion.
The ease of synthesis and chemical stability of simple, unmodified oligodeoxynucleotides has led to widespread investigation of their use as anti-sense reagents. This approach has been used with varying degrees of success in vitro against human immunodeficiency virus (HIV), Rous sarcoma virus, and c-myc oncogene expression, among others.28
Simple oligodeoxynucleotide anti-sense reagents may exert their effects in one or both of two ways. They can simply bind their target through duplex formation thereby reducing the available concentration of functional single-stranded target. Alternatively, in the case where the target is a single-stranded RNA, the RNA/DNA hybrid may Serve as a substrate for endogenous ribonuclease H (RNaseH). RNaseH is an enzyme which will cleave the RNA strand of an RNA/DNA hybrid through phosphodiester bond hydrolysis. The mediation of RNaseH can allow anti-sense reagents to operate at concentrations well below those required to drive all of the target to hybrid since the reagent itself is not cleaved and each molecule can direct the cleavage of many molecules of target.
A number of structural modification approaches to improve the function of oligodeoxynucleotides as anti-sense reagents have been investigated. One class of modification involves the attachment of chemical appendages to the reagent to stabilize the reagent/target duplex or cleave the target at the site of attachment. Acridine derivatives attached via flexible tethers have been shown to improve the thermodynamic stability of the duplex through intercalation5. Similarly, oligodeoxynucleotides bearing tethered psoralens can be covalently cross-linked to target following irradiation of the duplex6. Cross-linking can also be accomplished through the use of tethered alkylating agents7. Cleavage of the target through the use of oligodeoxynucleotides bearing tethered ethylenediaminetetraacetic acid (EDTA)/iron8 or 1,10-phenanthroline/copper9 has been demonstrated in vitro. Numerous other attachments for these purposes have been described. Functionalization with poly (L-lysine) has been employed to improve transport.31
The use of oligodeoxynucleotides as anti-sense reagents in vivo is hampered, however, by two fundamental problems. The first problem is that small single-stranded oligodeoxynucleotides are rapidly digested by endogenous nucleases. As a result high in vivo concentrations are difficult to sustain. The second problem is that oligodeoxynucleotides are highly charged having roughly one full negative charge per nucleotide residue. This generally results in a reduced rate of transport across membranes which in some cases limits the access to the ultimate site of action. These two effects combine to afford relatively low bioavailability.
The attachment of chemical functionalities (as described above) to the terminii of oligodeoxynucleotides can provide enhanced nuclease-resistance in some cases. Alpha-Oligodeoxynucleotides, in which the attachment of the base to the ribofuranosyl ring has been changed from beta- to alpha-, form parallel stranded duplexes and show increased nuclease resistance10.
In several approaches to solve the stability and transport problems, the central phosphorus atom in the linkage has been retained but attached atoms have been replaced or modified. O-Alkylphosphotriesters are uncharged but are more bulky than the natural linkage and show somewhat reduced chemical stability11,12. Phosphorothioate diesters are isostructural with the natural linkage and show increased resistance to nucleases but are still charged13. Methylphosphonates are uncharged and considerably more lipophilic but fully replaced hybrids are not substrates of RNaseH14.
One fundamental difficulty with these analogues is the fact that all derive from a single replacement of one of the non-bridging oxygen atoms on the phosphodiester linkage. Since that phosphorus is prochiral within the linkage, non-specific replacement of one oxygen results in the formation of a chiral center and hence a pair of diastereomers. Each additional non-specific replacement doubles the number of diastereomers present. These diastereomers have differing physical properties complicating analysis and in some cases have been shown to have widely differing abilities to form hybrids34. Thus oligodeoxynucleotide analogues with multiple linkage replacements are generally complex mixtures of species which can have widely differing biological efficacy.
The diastereomer mixture problem can be circumvented by replacement of both bridging oxygens in the phosphodiester linkage with the same chemical group. Along these lines, phosphorodithioate diesters have been investigated15. However, like the phosphorothioate linkage, this moiety is still charged.
Approaches involving more extensive modifications have also been reported. Replacement of the phosphodiester linkage with a carbon centered, neutral carbamate have been investigated16,17. The planarity of the carbamate linkage ensures that its introduction does not generate diastereomers but it represents a departure from tetrahedral geometry of the phosphodiester with consequences that have not been fully explored. At least one derivative has been shown to be able to form duplex with complementary target. The carbamate's nuclease sensitivity and ability to activate RNaseH have not yet been reported on.
Several recent studies have focused on oligodeoxynucleotides with partial phosphodiester linkage replacement. The rationale here is that one may be able to engineer a balanced profile of desirable properties (e.g., nuclease resistance, hybrid stability, RNaseH activation, etc.). These studies while useful are unlikely to lead to a general solution to the problems listed above.
With some analogues, the situation is further complicated by the observation that biological effects are being exerted in a non-sequence-specific manner18,19. The origins of these effects remain obscure.
The described list of modifications explored in attempts to enhance the function of oligodeoxynucleotides as anti-sense reagents is representative but is by no means exhaustive. Two recent reviews1,2 and one monograph32 dealing with this subject area are comprehensive.
A series of naturally occurring sulfamoyl mononucleoside antibiotics and some synthetic analogs have been described21-24. In addition, analogs of DNA containing sulfides, sulfoxides and sulfones as linking groups between subunits capable of forming bonds with natural oligonucleotides have been described.33 The β-decay of P32 oligonucleotides is expected to give a sulfate linkate oligonucleotide; however, such sulfate linked oligonucleotides have not been reported.
Applicants have developed a novel series of compounds in which one or more of the internucleotide phosphodiester linkages in oligonucleotide analogs have been replaced by a sulfur-based linkage. This linkage is isostructural and isoelectronic with the phosphodiester. Applicants have found the linkage to be synthetically accessible, chemically robust, nuclease resistant, and capable of supporting duplex formation. In addition, such compounds would have potential as antiviral agents and utility as hybridization probes.
There is provided by this invention a compound of the formula: ##STR1## A is H, OH, OR8, OQ or halogen; B is a naturally occurring nucleic acid base or synthetic modified nucleic acid base;
Y is RN or O;
Z is R7 N or O;
M is S(═O)═O, P(═O)--O-, P(═O)--S-, P(═S)--S-, P(═O)--OR3 , P(═O)--R9, P(═O)--SR4 or ##STR2## provided at least one M is S(═O)═O and when M is S(═O)═O, only one of Y and Z are O;
n is one or greater;
R is H or alkyl;
R1 is H2 PO3, H3 P2 O5, H4 P3 O7 and their suitable salts, H or a protecting group;
R2 is H2 PO3, H3 P2 O5, H4 P3 O7 and their suitable salts, H or a protecting group;
R3 is alkyl or cyanoethyl;
R4 is H or alkyl;
R5 is H or alkyl;
R6 is H or alkyl;
R7 is H or alkyl;
R8 is alkyl;
R9 is alkyl or cyanoethyl; and
Q is a protecting group.
In another aspect of this invention, intermediate compounds are provided: ##STR3## wherein: A is H, OH, OR2, OQ or halogen;
B is a naturally occurring nucleic acid base or synthetic modified nucleic acid base;
Y is RN or O;
Z is R1 N or O;
X is a suitable leaving group;
Q is a protecting group;
R is H or alkyl;
R1 is H or alkyl; and
R2 is alkyl or ##STR4##
In another aspect of this invention, a hybridization probe is provided.
FIG. 1. Demonstrates the melting profile of the modified oligodeoxynucleotides, 9a:9c, (circles) and the two oligodeoxynucleotides containing a single sulfamate linkage in each strand, 9b:9d, (closed squares). The buffer contained 200 mM sodium at pH 7.0.
FIG. 2. FIG. 2a shows an HPLC chromatogram of a standard mixture of dC, T, dG, and dA. FIG. 2b shows an HPLC chromatogram of the product mixture resulting from prolonged digestion: of oligodeoxynucleotide 9 c. The HPLC conditions used were as follows: C18 Rainin analytical column (4.6 mm�25 cm) with guard, flow of 1.0 ml/min, and a gradient of 10% water/methanol (solvent B) in 50 mM KH2 PO4 /water (solvent A) of 0-20 min=3.0% sol.B/sol.A), 20-40 min=3-40% sol.B/sol.A (linear), 40-45 min=40% sol.B/sol.A.
FIG. 3. This figure is a photograph of an autoradiogram of a 20% denaturing polyacrylamide gel illustrating cleavage of sulfamate or phosphate linked oligonucleotides by Nsi I.
FIG. 4. Demonstrates the meltings profile of d(AsA) vs. poly-uridine in a buffer of 10 mM magnesium H at pH 7.5.
All previous attempts to replace the phosphodiester linkage in oligodeoxynucleotide analogs for the purposes of enhancing function as anti-sense reagents represent partial solutions. No single phosphodiester replacement has a completely satisfactory profile of properties. Applicants have generated a list of chemical, biochemical, and structural criteria which, if met by a compound, are likely to lead to superior functions such as anti-sense, anti-viral and triplex functions. The linkage should be: 1) isostructural (isoelectronic and isosteric) with the phosphodiester; 2) nuclease resistant; 3) uncharged at physiological pH; 4) achiral; 5) chemically stable under physiological conditions; 6) synthetically accessible; and 7) amenable to analysis.
Applicants have developed compounds with at least one novel sulfur-centered linkages which meet all of these criteria. These linkages are sulfamate esters. Conceptually these derive from the sulfate diester which is isosteric and isoelectronic with the phosphodiester. Replacement of either bridging oxygen with a nitrogen affords a sulfamate ester. Applicants have found the internucleotide sulfamate linkage to be synthetically accessible, chemically robust, nuclease resistant, and capable of supporting duplex formation.
The novel compound of this invention is represented by the formula: ##STR5## A is H, OH, OR8, OQ or halogen; B is a naturally occurring nucleic acid base or synthetic modified nucleic acid base;
Y is RN or O;
Z is R7 N or O;
M is S(═O)═O, P(═O)--O-, P(═O)--S-, P(═S)--S-, P(═O)--OR3, P(═O)--R9, P(═O)--SR4 or ##STR6## provided at least one M is S(═O)═O and when M is S(═O)═O, only one of Y and Z are O;
n is one or greater;
R is H or alkyl;
R1 is H2 PO3, H3 P2 O5, H4 P3 O7 and their suitable salts, H or a protecting group;
R2 is H2 PO3, H3 P2 O5, H4 P3 O7 and their suitable salts, H or a protecting group;
R3 is alkyl or cyanoethyl;
R4 is H or alkyl;
R5 is H or alkyl;
R6 is H or alkyl;
R7 is H or alkyl;
R8 is alkyl;
R9 is alkyl or cyanoethyl; and
Q is a protecting group.
As far as the base (B) is concerned, no fundamental limitation is envisioned. One may include the naturally occurring nucleotide bases as well as synthetic modified nucleic acid bases such as inosine, deazaadenosine, etc. Preferably B is a residue of the naturally occurring nucleotide bases adenine, guanine, cytosine, thymine or uracil.30,33
With regard to the sugar, ribonucleotides, deoxyribonucleotides, and dideoxyribonucleotides are all possible. Sugars with halogen substitution (A is halogen) and altered sugar configuration (e.g., arabinosides and alpha-ribosides) may also be contemplated.30
In the above compound, n is at least one, but an n of up to 200 is accessible by standard methods. A compound of n greater than 200 could be made by ligation techniques.
With regard to the internucleotide linkage (M), Applicants have demonstrated the preparation of compounds with only a single sulfur-based linkage as well as the preparation of fully replaced compounds. Compounds where at least one of the sulfur based linkages of this invention is substantially near at least one terminus of the compound, are expected to be particularly useful. Such compounds will be substantially protected from exonuclease activity. Compounds that are not fully replaced with the sulfur based linkages comprise linkages as defined by M, which are as found in DNA, RNA as well as synthetic modified nucleotides known to those skilled in the art. It is understood that materials with any degree of partial replacement of phosphorous based linkages should be accessible by the methods applicants have developed. Furthermore, compounds with mixed ribose structures (e.g., single strands containing both ribonucleotides and deoxyribonucleotides) may be prepared.
Where R substituents are alkyl, alkyls of less than 5 carbon atoms are expected to be useful.
Additionally, one may attach the chemical appendages to the compounds of this invention for the purposes of stabilizing a duplex, crosslinking, or cleaving a target sequence or for facilitating transport(vide supra).5-9,31
Unlike the phosphodiester linkage, the sulfamate has directionality. The linkage may be oriented in one of two ways with respect to the compound terminii. The two forms will be referred to as 3'-NSO or 5'-NSO depending on whether the nitrogen is on the 3' or 5' side of the attached sulfur. ##STR7## Synthesis
Conceptually a disubstituted sulfamate can be prepared by the reaction of a hydroxy compound with a sulfamoyl-X [where X is a suitable leaving group] (Scheme 1) or by the reaction of an amino compound with alkoxysulfonyl-X (Scheme 2). Generally, the leaving group (X) is a group that is easily displaced (Examples include, halide, azide, sulfonates, etc.). Equivalent reactions with reduced sulfur reagents (e.g., sulfinyl groups) followed by oxidation may also be contemplated. ##STR8##
Both of these routes are feasible for the preparation of the sulfamate-linked oligonucleotide derivatives that are the subject of this application. Applicants have found one specific embodiment of Scheme 1 to be particularly efficacious. In this process, a 5'-N--(X-sulfonyl)-5'-amino-5'-deoxynucleoside intermediate is reacted with a hydroxy-bearing nucleoside or hydroxy-bearing oligonucleotide to afford a 3'-NSO sulfamate linked derivative. In this process, ##STR9## X is a suitable leaving group such as a halide, sulfonate, or azido group, with the azido group preferred. Similarly, 5'-NSO sulfamate linked derivatives may be prepared by reacting a 3'-N--(X-sulfonyl)-3'-amino-3'-deoxynucleoside with a hydroxy-bearing nucleoside or ##STR10## hydroxy-bearing oligonucleotide. These processes are generally carried out in the presence of a base but may also be facilitated by metal ions.
The key intermediates in these processes, the N--(X-sulfonyl)-amino-deoxynucleosides, may be conveniently prepared by the reaction of the appropriate amino-deoxynucleoside with a bifunctional sulfonating agent X-SO-2 -X'. For example, for X=azido, the N-(azidosulfonyl)-amino-deoxynucleoside is prepared by reacting the amino-deoxynucleoside with azidosulfonyl chloride.
These N--(X-sulfonyl)-amino-deoxynucleoside intermediates may also be reacted with amino-bearing nucleosides or amino-bearing oligonucleotides, to afford sulfamide-linked oligonucleotide derivatives that are also the subject of this application.
The intermediates of this invention are compounds of the formula: ##STR11## wherein: A is H, OH, OR2, OQ or halogen;
B is a naturally occurring nucleic acid base or synthetic modified nucleic acid base;
Y is RN or O;
Z is R1 N or O;
X is a suitable leaving group;
Q is a protecting group;
R is H or alkyl;
R1 is H or alkyl; and
R2 is alkyl or ##STR12##
The strategy for insertion of the sulfamate linkage into larger compounds of this invention depends on the degree of substitution desired. For introduction at a single site or just a few sites, a block-dimer approach is preferred. In this approach the standard chemistry of automated oligonucleotide synthesis based the stepwise addition of single nucleotides (3' to 5' growth) via nucleoside phosphoramidites is employed.39 At a selected point in the synthesis, a block-dimer phosphoramadite of this invention, a 5'-dimethyoxytrityl-protected, sulfamate-linked dinucleoside 3'-phosphoramidite is used in place of the normal reagent. This results in the addition of two nucleotides, linked by a sulfamate, to the 5'-terminus of the growing chain. The block multi-mer of this invention would allow n consecutive sulfamate linkages to be introduced.
The block multi-mer phosphoramidite compound of this invention is of the formula: ##STR13## A is H, OH, OR4, OQ, or halogen; B is a naturally occurring nucleic acid base or synthetic modified nucleic acid base;
n is at least one;
R1 is alkyl;
R2 is dialkylamino, morpholino, piperidino or pyrrolidono;
R3 is alkyl;
R4 is alkyl;
Y is RN or O;
Z is RN or O;
R is H or alkyl;
Q is a protecting group; and
V is a protecting group.
R, R1, R3 and R4 substituents with alkyls of less than 5 carbon atoms are expected to be useful.
V is a protecting group that can be cleaved under basic conditions, examples include cyanoethyl and methyl.
For all compounds of this invention, Q is alkyl or a protecting group possibly selected from acyl, trialkylsilyl, cyanoethyl, or a tetrahydropyran.
Where the A substituent contains an oxygen, three levels of protection can be imagined. In one case, the protecting group Q would be amenable to cleavage under the same conditions as for V. Another possibility is to make Q stable to the deprotection conditions used for removing V for stability reasons. The final case would be where Q is not to be removed at all, an alkyl group for example.
The issue of the stability of the sulfamate linkage is an important one in this approach. First, the linkage must be able to withstand repeated treatment under the cycling conditions: 1) removal of the 5'-trityl group with moderately strong acid; 2) coupling with a tetrazole activated phosphoramidite; and 3) oxidation with iodine under basic conditions. Applicants have found the internucleotide linkage to be fully stable to repeated treatments under these conditions. Secondly, the linkage must be able to stand up to the conditions required to cleave the nascent oligonucleotide from the resin and remove the protecting groups from the nucleotide bases. The base protection generally involves blocking the exocyclic amino groups through acylation (for C, A: benzoylation; for G: isobutyrylation). Both cleavage from the resin and base-deprotection are generally carried out by treating with concentrated aqueous ammonia. The cleavage from the resin occurs rapidly at room temperature but complete base deprotection requires more prolonged treatment at elevated temperatures. The sulfamate linkage is fully resistant to this prolonged treatment with concentrated aqueous ammonia.
Using the block dimer approach, one may synthesize compounds, oligonucleotide analogs, bearing from one to multiple sulfamate linkages. The only limitations are that the first linkage (on the 3'-terminus) may not be a sulfamate and one may not introduce consecutive sulfamates. (These limitations may be overcome, respectively, through the use of a resin linked sulfamate block dimer as a starter or through the use of block multimers.)
A different strategy is employed for the synthesis of compounds where all of the internucleotide linkages are to be replaced by the sulfamate. Here a stepwise synthesis is preferred. Synthesis is carried out in a 5'-3' direction adding a single nucleotide unit at a time. The 3'-protected-5'-azidosulfonamido monomer is activated in situ and added to the 3'-hydroxyl of the growing oligonucleotide.
This direction of chain-synthesis is the reverse of that employed in normal automated oligodeoxynucleotide synthesis. Thus the two processes are not trivially compatible. However, it is possible to conceive of cycles where either the sulfamate or the phosphodiester could be added as desired. For example, it should be possible using a 3'-azidosulfonamido-5'-protected monomer to incorporate sulfamate linkages at any point desired. This is a consequence of the extension direction now being 3' to 5', the same direction used in phosphoramidite extension.
In general an activated monomer approach is superior to a block dimer approach in that: a) all possible sequences can be addressed; and b) only four, as opposed to sixteen reagents are required.
The sulfamate-linked oligonucleotides may be useful as hybridization probes and as structural analogs for the study of protein/nucleic acid interactions.
Duplex Formation:
To function as an anti-sense reagent, the compound of this invention must retain the ability to recognize (hybridize to, anneal to, form duplex with) a complementary sequence. For short oligonucleotides, this may be conveniently assayed by observing the changes in UV absorption which accompany duplex formation. Generally, the absorption at 260 nm is measured as a function of temperature.
To study the effect of replacing a single phosphodiester linkage with the sulfamate on duplex formation, a pair of modified complementary 18-mers, each having a single sulfamate linkage, were compared to the corresponding pair of unmodified 18-mers. The two pairs showed virtually identical melting profiles (smooth S-curves) with the curve for the modified pair displaced to a slightly lower temperature. That the magnitude of the absorption change is identical and the curves are nearly identical in shape suggest that the duplex is perturbed very little by the sulfamate linkage and that it undergoes single-domain melting. The slightly lower transition temperature indicates that the modified duplex is slightly less stable than its unmodified counterpart under these conditions.
Attempts to demonstrate annealing of a T-homopolymer bearing all sulfamate linkages to a short dA homopolymer (with phosphodiesters) have been unsuccessful. This is somewhat perplexing but a similar phenomenon has been observed with carbamates and methylphosphonates: oligoTs (and Us) anneal poorly or not at all to polyA, yet sequences with other bases anneal well17,20. This may be a peculiarity of oligoTs when the linkage is uncharged.
The annealing of a sulfamate-linked adenosine dimer to polyU has been demonstrated. Here the interaction was very strong with a transition temperature some 10 degrees higher than the corresponding unmodified adenosine dimer.
Nuclease Resistance:
For stability in vivo, it is desirable that an anti-sense reagent be resistant to exonucleases, which digest from either terminus in a stepwise fashion, and endonucleases, which cleave in the middle of the chain.
To test for exonuclease resistance, several compounds of this invention having a single sulfamate linkage were subjected to prolonged treatment with a mixture of an exonuclease, snake venom phosphodiesterase, and alkaline phosphatase (to facilitate analysis). In each case, the sulfamate link was recovered intact (as the dimer) with the rest of the compounds fully digested to its constituent nucleosides.
Endonuclease resistance was tested by examining the cleavage of a short double stranded oligomer containing the Nsi 1 restriction endonuclease recognition sequence. This endonuclease normally cuts both strands within its recognition sequence in a staggered fashion. Replacement of the phosphodiester bond at the cleavage site in either or both Strands conferred resistance to cleavage in the altered strand (s).
A mixture of 8.9 g (24 mmol) of 5'-Azido-N6-benzoyl-2',5'-dideoxyadenosine (which can be prepared as in reference 36), 5.4 g (35 mmol) of t-butyldimethylsilyl chloride, and 3.3 g (48 mmol) of imidazole in 100 ml of anhydrous dimethylformamide (DMF) was stirred at room temperature (RT) for 20 hr. The solvent was removed under vacuum, and the residue was partitioned between one half saturated brine and EtOAc (2�200 ml). The combined organic layers were washed with brine, dried with sodium sulfate (dried), and evaporated (evap.). Purification by chromatography on silica (silica gel (240-400 mesh) from EM Science) using 20 to 40% EtOAc/DCM gave 11.1 g of 5'-Azido-N6-benzoyl-3'-O-t-butyldimethylsilyl-2',5'-dideoxyadenosine as a white foam: NMR (300 MHz,d6-DMSO) δ11.25 (br, 1H), 8.78 (s,1H), 8.71 (s, 1H), 8.05 (m, 2H), 7.5-7.7 (m, 3H), 6.52 (t, 1H,J=7 Hz), 4.72 (m, 1H), 4.02 (m,1H), 3.67 (dd,1H, J=13,7 Hz), 3.56 (dd,1H,J=13,5 Hz), 3.09 (m, 1H), 2.43 (m,1H), 0.92 (s,9H), 0.14 (s,6H).
A mixture of 5.08 g (10.4 mmol) of 5'-Azido-N6-benzoyl-3'-O-t-butyldimethylsilyl-2',5'-dideoxyadenosine and 2.1 g of 10% Pd/C in 69 ml of ethanol and 3.0 ml of acetic acid was stirred under 1 atm. of hydrogen for 4 hr. The slurry was filtered through celite washing with methanol p-Toluenesulfonic acid monohydrate (1.97 g, 10.4 mmol) was added and the mixture was agitated until dissolution. The solution was evap., and the resulting solid was chromatographed on silica (1 to 12% methanol/DCM) giving 4.63 g of 5'-Amino-N6-benzoyl-3'-O-t-butyldimethylsilyl-2',5'-dideoxyadenosine, p-toluenesulfonate salt: NMR (300 MHz, d6-DMSO) δ8.76 (s,1H), 8.74 (s,1H), 8.05 (d,2H, J=8 Hz ), 7.67 (t,1H,J=8 Hz), 7.57 (t,2H,J=8 Hz), 7.48 (d,2H,J=8 Hz), 7.11 (d,2H,J=8 Hz), 6.53 (t,1H,J=7 Hz), 4.68 (m,1H), 4.01 (m,1H), 3.06 (m,2H), 2.56 (m,1H), 2.39 (m,1H), 2.29 (s,3H), 0.93 (s,9H), 0.15 (s,6H).
A mixture of 4.24 g (6.68 mmol) of 5'-amino-N6-benzoyl-3'-O-t-butyldimethylsilyl-2',5'-dideoxyadenosine and 4 Å molecular sieves (˜5 g) in 10 ml of anhydrous acetonitrile and 20 ml of anhydrous DCM was stirred under nitrogen for 2 hr. After adding 0.93 ml (6.68 mmol) of triethylamine, the mixture was stirred for 5 min, and 0.42 ml of chlorosulfonylazide (1M in acetonitrile) were added. After stirring the reaction for 1 hr., the slurry was filtered and the filtrate was partitioned between water and EtOAc (2�100 ml). The combined organic layers were washed with brine, dried, and evap. Purification using chromatography on silica (1 to 3% MeOH/DCM) gave 0.89 g of N6-benzoyl-3'-O-t-butyldimethylsilyl-5'-sulfamoylazido-2',5'-dideoxyadenosine as a white foam: NMR (300 MHz,d6-DMSO) δ11.22 (br,1H), 9.28 (br,1H), 8.75 (s,1H), 8.71 (s,1H), 8.05 (d,2H, J=8 Hz), 7.66 (t,1H,J=8 Hz), 7.56 (t,2H,J=8 Hz), 6.52 (t,1H,J=8 Hz), 4.67 (m,1H), 4.00 (m,1H), 3.07 (m,1H), 2.34 (m,1H), 0.93 (s,9H), 0.15 (s,6H). IR (KBr) 3400, 3080 2950, 2930, 2880, 2860, 2130, 1700, 1610, 1585, 1510, 1370, 1170 cm-1.
A mixture of 12.09 g (35.1 retool) of 5'-Azido-N6-benzoyl-2',5'-dideoxycytidine (which can be prepared as in reference 36), 7.94 g (52.7 mmol) of t-butyldimethylsilyl chloride, and 4.78 g (70.3 mmol) of imidazole were stirred in 70 ml of anhydrous DMF under nitrogen at RT for 20 hr. The DMF was removed under vacuum, and the resulting mixture was dissolved in DCM and loaded directly on a column of silica. A 25% EtOAc/DCM solution was used to elute the product from the column. Evap. of the fractions containing the product gave 13.35 g of 5'-Azido-N6-benzoyl-3'-O-t-butyldimethylsilyl-2',5'-dideoxycytidine as a white foam: NMR (300 MHz,d6-DMSO) δ11.31 (br,1H), 8.18 (d,1H,J=8 Hz), 8.01 (m,2H), 7.63 (m,1H), 7.56 (t,2H,J=7 Hz), 7.39 (d,1H,J=8 Hz), 6.20 (t,1H,J=7 Hz), 4.38 (m,1H), 3.96 (m,1H), 3.68 (dd,1H,J=13,6 Hz), 3.61 (dd,1H,J=13,5 Hz), 2.30 (m,1H), 0.88 (s,9H), 0.10 (s,6H).
A mixture of 6.88 g (15 mmol) of 5'-Azido-N6-benzoyl-3'-O-t-butyldimethylsilyl-2',5'-dideoxycytidine and 3.0 g of 10% Pd/C in 130 ml of ethanol and 5 ml of acetic acid was stirred under 1 atm. of hydrogen for 4 hr. The slurry was filtered through celite washing with methanol. p-Toluenesulfonic acid monohydrate (2.85 g, 15 mmol) was added and the mixture was agitated until dissolution. The solution was evap., and the resulting solid was chromatographed on silica (1 to 12% methanol/DCM) giving 6.32 g of pure 5'-amino-N6-benzoyl-3'-O-t-butyldimethylsilyl-2',5'-dideoxycytidine p-toluenesulfonate salt as a tan solid: NMR (300 MHz,d6-DMSO) δ8.31 (d,1H,J=8 Hz), 8.02 (d,2H,J=8 Hz), 7.64 (m,1H), 7.52 (m,4H), 7.38 (d,1H,J=8 Hz), 7.12 (d,2H,J=8 Hz), 6.19 (t,1H,J=6.5 Hz), 4.43 (m,1H), 3.97 (m,1H), 3.02 (m,2H), 2.30 (m,5H), 0.89 (s,9H), 0.11 (s,6H).
A mixture of 3.40 g (5.63 mmol) of 5'-amino-N6-benzoyl-3'-O-t-butyldimethylsilyl-2',5'-dideoxycytidine p-toluenesulfonate salt and 4 Å molecular sieves (˜5 g) in 30 ml of 50% anhydrous DCM/acetonitrile was stirred under nitrogen for 1 hr After cooling to 0� C., 0.78 ml (5.63 mmol) of triethylamine was added and stirring was continued for 10 min. Finally 3.9 ml (3.9 mmol) of chlorosulfonylazide (1M in acetonitrile) was added, the cold bath was removed, and the reaction was stirred for 2.5 hr. The slurry was filtered, and the filtrate was partitioned between one half saturated brine and 300 ml of EtOAc. The organic layer was washed with brine, dried, and evap. Purification by chromatography on silica (1 to 4% methanol/DCM) gave 0.99 g of product that contained two impurities. Furthur chromatography afforded pure N6-benzoyl-3'-O-t-butyldimethylsilyl-5'-sulfamoylazido-2',5'-dideoxycytidine as a white foam: NMR (300 MHz,d6-DMSO) δ11.27 (br,1H), 9.30 (br,1H), 8.21 (d,1H,J=8 Hz), 8.01 (m,2H), 7.62 (m,1H), 7.50 (t,2H,J=8 Hz), 7.37 (d,1H,J=8 Hz), 6.17 (t,1H,J=6.5 Hz), 4.39 (m,1H), 3.95 (m,1H), 3.35 (m,2H), 2.27 (m,2H), 0.89 (s,9H), 0.10 (s,6H).
Preparation of 3'-O-t-butyldimethylsilyl-N4-i-butyryl-O6- (4-nitrophenethyl)-5'--sulfamoylazido-2',5'-dideoxyguanosine
Using the procedure in reference 37, 5.00 g (17.5 mmol) of 2'-deoxyguanosine (Sigma, St. Louis, Mo.), 63 ml of anhydrous DCM, 13 ml of anhydrous pyridine, and 9.3 ml (88 mmol) of isobutyryl chloride gave 10.34 g of crude N4, (3',5'-O-)-tri-i-butyryl-2'-deoxyguanosine.
After dissolving 10.34 g (˜17 mmol) of crude N4, (3',5'-O-)-tri-i-butyryl-2'-deoxyguanosine in 70 ml of anhydrous DCM under nitrogen, 8.91 g (34 mmol) of triphenylphosphine and 5.68 g (34 mmol) of 4-nitrophenethyl alcohol were added and dissolved with stirring After cooling to 0� C., 5.3 ml (34 mmol) of diethyldiazodicarboxylate were added over 5 minutes (min). The cold bath was removed, and the reaction was stirred for 18 hr. A TLC of the reaction mixture (10% methanol/DCM) indicated some of the starting material was still present. Triphenylphosphine (4.45 g), 4-nitrophenethyl alcohol (5.30 g), and diethyldiazodicarboxylate (2.65 ml) were added sequentially, and the mixture was stirred another one hr. The solvent was evaporated, and the residue was used directly in the next reaction. A sample was purified by chromatography on silica (1 to 5% methanol/DCM) to give N4, (3',5'-O-tri-i-butyryl-O6-(4-nitrophenethyl)-2' -deoxyguanosine as a white foam: NMR (300 MHz, CdCl3) δ8.17 (d,2H,J=8 Hz), 8.07 (br, 1H), 7.97 (s, 1H), 7.53 (d, 2H, J=8Hz), 6.36 (dd, 1H, J=7,6 Hz), 5.43 (m, 1H), 4.84 (t,2H, J=7 Hz), 4.54 (dd, 1H, j=12,5 Hz), 4.39 (m, 2H), 3.33 (t,2H, J=7 Hz), 2.5-3.1 (m,5H), 1.39 (d,6H,J=7 Hz), 1.32 (d,6H,J=7 Hz), 1.26 (d,3H,J=7 Hz), 1.24 (d,3H,J=7 Hz);
Crude N4, (3',5═-O-) -tri-i-butyryl-O6-(4-nitrophenethyl)-2'-deoxyguanosine (˜17 mmol) was dissolved in 400 ml of methanol and 400 ml of 27% ammonium hydroxide were added. The mixture was cloudy so enough methanol was added (100 ml) to make the reaction homogeneous. After stirring for 20 hr at RT, the solvents were evap. and the residue was taken up in 100 ml of DCM. Enough hexane was added to make the solution cloudy. After standing overnight, the light yellow crystals were collected to give 5.83 g of N4-i-butyryl-O6-(4-nitrophenethyl)-2'-deoxyguanosine. A second crystallization gave an additional material for a total of 8 .60 g of N4-i-butyryl-O6-(4-nitrophenethyl)-2'-deoxyguanosine: NMR (300 MHz, 20% d4-methanol/CDCl3) δ8.18 (d,2H,J=9 Hz), 8.17 (s,1H), 7.57 (d,2H,J=9 Hz), 6.38 (t,1H,J=7 Hz), 4.84 (t,2H,J=7 Hz), 4.72 (m,1H), 4.07 (q,1H,J=4 Hz), 3.83 (ddd,2H,J=33,12,4 Hz), 3.33 (t,2H,J=7 Hz), 2.75-2.9 (m,2H), 2.44 (ddd,1H,J=14,7,4 Hz), 1.37 (d,6H,J=8 Hz).
A solution of 7.28 g (14.9 mmol) of N4-i-butyryl-O6-(4-nitrophenethyl)-2'-deoxyguanosine in 75 ml of anhydrous pyridine was cooled to 0� C. under nitrogen. After adding 2.84 g (14.9 mmol) of p-toluenesulfonyl chloride, the reaction was stirred at 0� C. for 2 hr and then at 5� C. for 18 hr. The pyridine was evap., and the residue was partitioned between water and EtOAc. The organic layer was washed with brine, dried, and evap. to give 6.22 g. Purification by chromatography on silica (1 to 5% methanol/DCM) gave 4.08 g of N4-i-butyryl-O6-(4-nitrophenethyl)-5'-O-p-toluenesulfonyl-2'-deoxyguanosine as a white foam: (300 MHz,20% d6-DMSO) δ10.34 (br,1H), 8.24 (s,1H), 8.19 (d,2H,J=8 Hz), 7.67 (d,2H,J=8 Hz), 7.61 (d,2H,J=8 Hz), 7.22 (d,2H,J=8 Hz), 6.38 (t,1H,J=7 Hz), 5.46 (d,1H,J=5 Hz), 4.82 (t,2H,J=7 Hz), 4.64 (m,1H), 4.33 (ddd,2H, J=26,12,8 Hz), 3.97 (m,1H), 3.35 (t,2H,J=7 Hz), 2.82 (m,2H), 2.27 (s,3H)i, 1.12 (d,3H,J=7 Hz), 1.10 (d,3H,J=7 Hz).
A solution of 4.08 g (6.35 mmol) of N4-i-butyryl-O6-(4-nitrophenethyl)-5'-O-p-toluenesulfonyl-2'-deoxyguanosine and 467 mg (9.52 mmol) of lithium azide (Kodak, Rochester, N.Y.) in 21 ml of anhydrous DMF was heated to 100� C. for 3 hr. The solvent was removed under vacuum. The residue was partitioned between water and EtOAc. The organic layer was washed with brine, dried, and evap. to give 3.95 g of crude product. Purification by chromatography on silica (1 to 10% methanol/DCM) gave 1.47 g of the less polar material and 419 mg of the more polar material. The less polar material was a white foam and was found to be 5'-azido-N4-i-butyryl-O6-(4-nitrophenethyl)-2',5'-dideoxyguanosine: NMR (300 MHz, d6-DMSO) δ10.40 (br,1H), 8.41 (s,1H), 8.18 (d,2H,J=8 Hz), 7.64 (d,2H,J=8 Hz), 6.37 (t, 1H,J=7 Hz), 5.46 (d,1H,J=4 Hz), 4.79 (t,2H,J=7 Hz), 4.63 (m,1H), 3.97 (m,1H), 3.86 (dd,1H,J=13,8 Hz), 3.53 (dd,1H,J=13.5 Hz), 3.36 (t,2H,J=7 Hz), 3.05(m,1H), 2.84 (m,1H), 2.28 (ddd,1H,J=14,6,3 Hz), 1.12 (d,6H,J=8 Hz).
In the same manner as for 5'-Azido-N6-benzoyl-3'-O-t-butyldimethylsilyl-2',5'-dideoxycytidine, 1 47 g (2 87 mmol) of 5'-azido-N4-i-butyryl-O6- (4-nitrophenethyl)-2',5'-dideoxyguanosine, 0 65 g (4.3 mmol) of t-butyldimethylsilyl chloride, 0.39 g (5.7 mmol) of imidazole, and 10 ml of anhydrous DMF was used to obtain after chromatography on silica (10% EtOAc/DCM) 1.49 g of 5'-azido-3'-O-t-butyldimethylsilyl-N4-i-butyryl-O6-(4-nitrophenethyl)-2'-dideoxyguanosine as a white foam: NMR (300 MHz, d6-DMSO) δ10.40 (br,1H), 8.40 (s,1H), 8.18 (d,2H,J=9 Hz), 7.65 (d,2H,J=9 Hz), 6.35 (t,1H,J=7 Hz), 4.78 (t,2H,J=7 Hz), 4.73 (m,1H), 3.94 (m,1H), 3.84 (dd,1H,J=13,8 Hz), 3.56 (dd,1H,J=13,5 Hz), 3.31 (t,2H,J=7 Hz), 3.10 (m,1H), 2.82 (m,1H), 2.29 (m,1H), 1.10 (d,6H,J=7 Hz), 0.89 (s,9H), 0.14 (s,3H), 0.12 (s,3H).
A mixture of 100 mg (0.159 mmol) of 5'-azido-3'-O-t-butyldimethylsilyl-N4-i-butyryl-O6-(4-nitrophenethyl)-2',5'-dideoxyguanosine, 0.064 ml (0.64 mmol) of 1,3-propanedithiol, 0.089 ml (0.64 mmol) of triethylamine, and 1 ml of methanol was stirred under nitrogen at RT for 4 days. After adding 0.5 ml of acetic acid, the reaction was concentrated under vacuum. Purification by chromatography on silica (1 to 15% methanol/DCM) gave 121 mg of the acetic acid salt of the product. This material was partitioned between saturated potassium carbonate and EtOAc. The organic layer was dried and evap to give 91 mg of 5'-amino-3'-O-t-butyldimethylsilyl-N4-i-butyryl-O6-(4-nitrophenethyl)-2',5'-dideoxyguanosine: NMR (300 MHz, CDCl3) δ8.17 (d,2H,J=8 Hz), 7.98 (s,1H), 7.90 (br,1H), 7.52 (d,2H,J=8 Hz), 6.28 (t,1H,J= 7 Hz), 4.80 (t,2H,J=7 Hz), 4.71 (m,1H), 3.93 (q,1H,J=4 Hz), 3.32 (t,2H,J=7 Hz), 3.04 (dd,1H,J=14,4 Hz), 2.93 (dd,1H,J=14,6 Hz), 2.84 (m,1H), 2.39 (m,1H), 1.28 (d,6H,J=7 Hz), 0.93 (s,9H), 0.13 (s,3H), 0.11 (s,3H).
A solution of 90 mg (0.15 mmol) of 5'-amino-3'-O-t-butyldimethylsilyl-N4-i-butyryl-O6-(4-nitrophenethyl)-2',5'-dideoxyguanosine in 2 ml of anhydrous DCM was stirred at RT under nitrogen while 0.11 ml of chlorosulfonylazide (1M in acetonitrile) was added. After stirring for 1 hr., the solution was partitioned between one half saturated brine and EtOAc. The organic layer was washed with brine, dried, and evap. The residue was purified by chromatography on silica (1 to 3% methanol/DCM) to give 18 mg of 3'-O-t-butyldimethylsilyl-N4-i-butyryl-O6-(4-nitrophenethyl)-5'-sulfamoylazido-2',5'-dideoxyguanosine: NMR (300 MHz, CDCl3) δ8.67 (br,1H), 8.46 (t,1H,J=6 Hz), 8.16 (d,2H,J=8 Hz), 7.81 (s,1H), 7.54 (d,2H,J=8 Hz), 6.17 (dd,1H,J=8,6 Hz), 4.87 (m,3H), 4.17 (m,1H), 3.57 (m,1H), 3.34 (t,2H,J=7 Hz), 2.98 (m,1H), 2.65 (m,1H), 2.25 (m,1H), 1.27 (d,3H,J=7 Hz), 1.26 (d,3H,J=7 Hz), 0.93 (s,9H), 0.16 (s,3H), 0.14 (s,3H).
A solution of 25.0 g (93.6 mmol) of 5'-azido-5'-deoxythymidine (which can be prepared as in reference 36), 21.1 g (140 mmol) of t-butyldimethylsilyl chloride, and 12.7 g (187 mmol) imidazole in 190 ml of anhydrous DMF was stirred at RT under nitrogen for 18 hr. The DMF was removed under vacuum. The oil was partitioned between ether (500 ml) and 1N NaOH (2�300 ml). The aqueous layers were cooled with ice, and 650 ml of 1N HCl were added. The product was extracted with EtOAc (2�750 ml), and the organic layer was washed with brine, dried, and evap. After remaining under high vacuum for 24 hr, the oil crystallized to give 27.9 g of 5'-azido-3'-O-t-butyldimethylsilyl-5'-deoxythymidine as a white solid: NMR (300 MHz,d6-DMSO) δ11.34 (br,1H), 7.50 (s,1H), 6.16 (t,1H,J=7 Hz), 4.36 (m,1H), 3.84 (m,1H), 3.55 (m,2H), 2.34 (m,1H), 2.05 (m,1H), 1.79 (s,3H), 0.86 (s,9H), 0.08 (s,6H).
A mixture of 15.0 g (39.3 mmol) of 5'-azido-3'-O-t-butyldimethylsilyl-5'-deoxythymidine and 4.0 g of 10% Pd/C in 200 ml of EtOAc was stirred vigorously under 1 atm. of hydrogen for 4.5 hr. The slurry was filtered through celite, rinsing with methanol. The filtrate was evap. to give 15.5 g of a thick oil (contains some solvent). A sample was purified by chromatography on silica (2-10% methanol/DCM) to give pure 5'-amino-3'-O-t-butyldimethylsilyl-5═-deoxythymidine as a white solid: NMR (300 MHz, d6-DMSO) δ7.64 (s,1H), 6.13 (dd,1H,J=8,6 Hz), 4.37 (m,1H), 3.65 (m,1H), 2.78 (m,2H), 2.21 (m,1H), 2.01 (ddd,1H,J=12,6,4 Hz), 1.79 (s,3H), 0.88 (s,9H), 0.08 (s,6).
A mixture of 5.52 g of 5'-amino-3'-O-t-butyldimethylsilyl-5'-deoxythymidine and 168 mg of ammonium sulfate in 150 ml of hexamethyldixilazane (HMDS) was heated to dissolution under nitrogen. After stirring at RT for 18 hr, the slurry was heated to 80� C. for 2 hr. The HMDS was removed under vacuum to give a thick oil which was dissolved in 45 ml of anhydrous acetonitrile under nitrogen. This solution was added to an addition funnel and dripped into 10 ml of chlorosulfonylazide (1M in acetonitrile) precooled to 0� C. over 25 minutes. After the addition, the cold bath was removed, and the mixture was stirred for 1 hr. The reaction was poured onto 200 ml of half saturated brine and extracted with EtOAc (2�200 ml). The combined organic layers were washed with brine, dried, and evap. to give 5.29 g of a solid. Purification by chromatography on silica (1 to 5% methanol/DCM) gave 3.70 g of reasonably pure 3'-O-t-butyldimethylsilyl-5'-sulfamoylazido-5'-deoxythymidine as a tan foam: NMR (300 MHz,CDCl3) δ8.57 (br,1H), 7.02 (s,1H), 6.88 (br,1H), 5.75 (t,1H,J=8 Hz), 4.49 (m,1H), 4.04 (m,1H), 3.48 (m,2H), 2.72 (m,1H), 2.18 (m,1H), 1.92 (s,3H), 0.89 (s,9H), 0.11 (m,6H).
A mixture of 1.00 g (3.53 mmol) of 5'-amino-3'-O-acetyl-5'-deoxythymidine (which can be prepared as in reference 38) and 94 mg of ammonium sulfate was stirred in 20 ml of HMDS under nitrogen at RT for 18 hr. The slurry was gently heated (60� C.) for 24 hr. The HMDS was removed under vacuum into a trap at -78� C. Acetonitrile (40 ml) was added under nitrogen giving a homogenous solution. After adding 5.0 ml of chlorosulfonylazide (1.0M in acetonitrile) to the reaction, the mixture was stirred for 1.5 hr. Water (50 ml) was added and the product was extracted with EtOAc (2�150 ml). The organic layers were washed with brine, dried, and evap. giving 0.89 g of a solid. Purification by chromatography on silica (1 to 5% methanol/DCM) gave 0.64 g of pure 3'-O-acetyl-5'-sulfamoylazido-5'-deoxythymidine as a foam: NMR (300 MHz, d6-DMSO) δ11.40 (Br,1H), 9.31 (Br,1H), 7.59 (s,1H), 6.17 (dd,1H,J=7.5,6.5 Hz), 5.14 (m,1H), 4.03 (m,1H), 3.44 (m,2H), 2.47 (m,1H), 2.25 (m,1H), 2.07 (s,3H), 1.80 (s,3 H); IR (film) 3200, 2920, 2840, 2135, 1730, 1685, 1365, 1240, 1170 cm-1; Mass Spectrum (FAB) meas 388.98 m/z, calcd for C12H16N6O7S 389.08 (M+H);
A slurry of 2.17 g of 3'-azido-3'-deoxythymidine (Aldrich, Milwaukee, Wis.) and 0.91 g of 10% Pd/C in 25 ml of ethanol were vigorously stirred under 1 atm. of hydrogen for 4 hr. The mixture was filtered through celite, washed with methanol, and the filtrate was evap. Purification by chromatography on silica (2 to 40% methanol/DCM) gave 1.72 g of 3'-amino-3'-deoxythymidine as a light yellow solid: NMR (300 MHz,d6-DMSO) δ7.76 (s,1H), 6.07 (t,1H,J=6 Hz), 4.97 (m,1H), 3.5-3.7 (m,3H), 3.39 (q,1H,J=7 Hz), 3.33 (br,2H), 1.9-2.15 (m,2H), 1.77 (s,3H).
A solution of 1.04 g of 3'-amino-3'-deoxythymidine, 0.90 ml of triethylamine, and 1.5 ml of ethyl trifluoroacetate in 10 ml of methanol was stirred under nitrogen at RT for 20 hr. Evaporation gave a product that was used without purification. A sample was subjected to column chromatography on silica (1 to 20% methanol/DCM) affording pure 3'-trifluoroaceamido-3'-deoxythymidine: NMR (300 MHz,d6-DMSO) δ11.33 (br,1H), 9.84 (br,1H), 7.76 (s,1H), 6.26 (t,1H,J=7 Hz), 5.15 (t,1H,J=5 Hz), 4.47 (m,1H), 3.89 (m,1H), 3.6 (m,2H), 2.27 (m,2H), 1.78 (s,3H).
To a solution of crude 3'-trifluoroaceamido-3'-deoxythymidine, in pyridine was added 1.2 ml of triethylamine, 0.10 g of N,N-dimethylaminopyridine, and 2.2 g of dimethoxytrityl chloride under nitrogen. After stirring for 18 hr., the solvent was evap. The dark oil was partitioned between one half saturated bicarbonate and EtOAc. The organic layer was washed with brine, dried, and evap. Purification by chromatography on silica (1 to 7% methanol/DCM) gave 2.18 g of pure 5'-dimethoxy-trityl-3'-trifluoroacetamido-3'-deoxythymidine as a yellow solid: NMR (300 MHz, CDCl3) δ8.89 (br,1H), 8.31 (d,1H,J=7 Hz), 7.65 (s,1H), 7.2-7.45 (m,9H), 6.84 (d,4H,J=9 Hz), 6.51 (t,1H,J=7 Hz), 4.72 (m,1H), 4.09 (m,1H), 3.78 (s,6H), 3.53 (m,2H), 2.45 (m,2H), 1.64 (s,3H).
A solution of 1.54 g of 5'-dimethoxytrityl-3'-trifluoroacetamido-3'-deoxythymidine in methanol was saturated with ammonia gas at RT. The reaction vessel was sealed for 4 days. After the solvent was evap., the residue was purified using chromatography on silica (1 to 10% methanol/DCM) giving 1.17 g of 3'-amino-5'-O-dimethoxytrityl-3'-deoxythymidine: NMR (300 MHz,d6-DMSO) δ7.51 (s,1H), 7.2-7.45 (m,9H), 6.88 (d,4H,J=8 Hz), 6.14 (t,1H,J=6 Hz), 3.74 (s,6H), 3.68 (m,1H), 3.50 (q,1H,J=7 Hz), 3.33 (br,2H), 3.21 (m,2H), 2.21 (m,1H), 2.03 (m,1H), 1.47 (s,3H).
In the same manner as with example 4, 605 mg of 3'-amino-5-O-dimethoxytrityl-3'-deoxythymidine, 20 ml of HMDS, 0.78 ml of chlorosulfonylazide (1M in acetonitrile), nitrile), and 6 ml of anhydrous acetonitrile were used to obtain (after purification on silica using 1 to 5% methanol/DCM) 217 mg of 5'-dimethoxytrityl-3'-sulfamoylazido-3'-deoxythymidine as a light orange solid: NMR (300 MHz,CDCl3) δ7.58 (s,1H), 7.2-7.4 (m,9H), 6.85 (d,4H,J=9 Hz), 6.55 (t,1H,J=7 Hz), 4.31 (m,1H), 4.23 (m,1H), 3.78 (s,6H), 3.50 (dd,1H,J=11,3 Hz), 3.39 (dd,1H,J=11,3 Hz), 2.46 (m,2H), 1.47 (s,3H).
Stirred a mixture of 480 mg of the compound from Example 4 and 493 mg of N6-benzoyl-5'-dimethoxytrityl-2'-deoxyadenosine (Aldrich, Milwaukee, Wis.) in 7 ml of dry acetonitrile with some 4 Å mol. sieves for 2 hr. Added 0.17 ml of triethylamine to the reaction and stirred 18 hr. The mixture was filtered and evap. Purification by chromatography on silica (1-7% methanol/DCM) gave 621 mg of (DMTO)ABz sT(OTBDMS) as a white solid: NMR (300 MHz, d6-DMSO) δ11.33 (s,1H), 11.23 (br,1H), 8.58 (s,1H), 8.57 (s,1H), 8.51 (br,1H), 8.05 (m,2H), 7.5-7.7 (m,4H), 7.34 (m,2H), 7.21 (m,7H), 6.80 (dd,4H,J=8,5 Hz), 6.52 (t,1H,J=7 Hz), 6.14 (t,1H,J=7 Hz), 5.28 (m, 1H), 4.37 (m,2H), 3.81 (m,1H), 3.71 (s,6H), 3.29 (m,4H), 3.14 (dd,1H,J=17,5 Hz), 2.78 (m,1H), 2.26 (m,1H), 2.04 (m,1H), 1.77 (s,3H), 0.87 (s,9H), 0.09 (s,6H). mass spectrum (FAB) m/z 1075.47 (assigned M+H), calcd for C54H62N8O12SSi+H 1075.40. IR (CH2C12) 3380, 3180, 3060, 2960, 2930, 2860, 1695, 1610, 1585, 1510, 1360, 1180, 1250 CM-1.
The fully protected dimer, (DMTO) ABz sT(OTBDMS), was treated with a total of 6 ml of 1M tetra-n-butylammonium fluoride/THF (Aldrich) in four equal aliquots in one hr. intervals (small s designates sulfamate linkage). After the final additon, the reaction was stirred for 1 hr. The solution was partioned between 1M sodium dihydrogen phosphate and ethyl acetate (EtOAc). The organic layer was washed with brine, dried, and evap. The crude product was chrom. on silica (1-7% methanol/DCM) gaving 420 mg of (DMTO)ABz sT(OH) as a solid: NMR (300 MHz,d6-DMSO)�11.31 (Br,1H), 11.22 (br,1H), 8.58 (s,2H), 8.47 (br,1H), 8.03 (m,2H), 7.45-7.7 (m,4H), 7.34 (m,2H), 7.20 (m,7H), 6.81 (dd,4H,J=8,5 Hz), 6.52 (t,1H,J=7 Hz), 6.14 (t,1H,J=7 Hz), 5.39 (d,1H,J=5 Hz), 5.29 (m,1H), 4.38 (m,2H), 4.17 (m,1H), 3.80 (m,1H), 3.72 (s,6H), 3.3 (m,4H), 3.14 (m,1H), 2.79 (m,1H), 2.4 (m,1H), 2.15 (m,1H), 1.76 (s, 3H). IR (KBR) 3400, 3180, 3055, 2955, 930, 2830, 1690, 1610, 1580, 1510, 1360, 1250, 1180 cm-1. mass spectrum (FAB) 961.66 (M+H), calcd for C48H48N8O12S+H 961.07.
To a solution of 102 mg of (DMTO)ABz sT(OH) in 5 ml of acetonitrile was added 0.22 ml of dichloroacetic acid. The orange solution was stirred at RT for 15 min. After pouring the reaction into one half saturated bicarbonate, the product was extracted with EtOAc (2�40ml). The combined organic layers were washed with brine, dried, and evap. Purification by chromatography on silica (1 to 10% methanol/DCM) gave 68 mg of (HO)ABz sT(OH): NMR (300 MHz, d6-DMSO) δ11.33 (br,1H), 11.26 (br,1H), 8.77 (s,1H), 8.70 (s,1H), 8.47 (br,1H), 8.06 (d,2H,J=8 Hz), 7.66 (t,1H,J=8 Hz), 7.59 (s,1H), 7.55 (d,2H,J=8 Hz), 6.52 (t,1H,J=7 Hz), 6.18 (t,1H,J=7 Hz), 5.41 (d,1H,J=5 Hz), 5.32 (t,1H,J=5 Hz), 5.23 (m,1H), 4.25 (m,1H), 4.19 (m,1H), 3.82 (m,1H), 3.65 (m,2H), 3.0-3.3 (m,3H), 2.78 (m,1H), 2.0-2.3 (m,2H), 1.79 (s,3H). IR (KBr) 3420, 1690, 1620, 1585, 1360, 1175 cm-1. mass spectrum (FAB) meas. 659.23 (M+H), calcd for C27H30N8O10S+H: 659.19.
A slurry of 31 mg of (HO)ABz sT(OH) in 3 ml of methanol was saturated at RT with ammonia gas. The reaction was sealed and heated to 55� C. for 4 hr. The solvent was evap. Purification by chromatography on silica (methanol/DCM gradient) gave 19 mg of d(AsT): NMR (300 MHz, d6-DMSO) δ [partial spectrum] 8.34 (s,1H), 8.14 (s,1H), 7.35 (s,1H), 7.41 (br,2H), 6.37 (dd,1H,J=9,6 Hz), 6.17 (t,1H,J=7 Hz), 5.67 (br,1H), 5.39 (d,1H,J=5 Hz), 5.18 (m,1H), 4.2 (m,2H), 3.81 (m,1H), 3,63 (m,2H), 3.19 (m,1H), 3.01 (m,1H), 2.66 (m,1H), 2.18 (m,1H), 1.78 (s,3H).
A mixture of 57 mg (0.10 mmol) of the compound from Example 1, 65 mg (0.10 mmol) and N6-benzoyl-5'-dimethoxytrityl-2'-deoxyadenosine (Aldrich) in 0.5 ml of acetonitrile was stirred at RT under nitrogen. Triethylamine 0.028 ml (0.2 mmol) was added and the reaction was stirred for 20 hr. After evaporation of the mixture and purification on silica (1-3% methanol/DCM) 87 mg of the fully protected dimer, (DMTO)ABz sABz (OTBDMS), were obtained: NMR [partial spectrum] (300 MHz,d6-DMSO) δ11.23 (br,2H), 8.66 (s,1H), 8.65 (s,1H), 8.58 (s,1H), 8.56 (s,1H), 8.56 (br,1H), 8.04 (d,4H,J=8 Hz), 7.5-7.7 (m,6H), 7.15-7.35 (m,9H), 6.77 (dd,4H,J=8,6 Hz), 6.53 (t,1H,J=7 Hz), 6.47 (t,1H,J= 7 Hz), 5.27 (m,1H), 4.64 (m,1H), 4.34 (m,1H), 4.01 (m,1H), 3.68 (s,6H), 3.02 (m,1H), 2.78 (m,1H), 2.36 (m,1H), 0.91 (s,9H), 0.15 (s,6H).
51 mg of (DMTO)ABz sABz (OTBDMS) were treated under nitrogen with a total of 1.5 ml of 1M tetra-n-butylammonium fluoride in THF added in three aliquots over 1 hr intervals. The reaction was stirred at RT for 1 hr. after the final addition of the fluoride reagent. The reaction was partitioned between 1M sodium dihydrogen phosphate and EtOAc. The organic layer was washed with brine, dried, and evap. Purification by column chromatography on silica (1-6% methanol/DCM) gave 36 mg of pure (DMTO) d(ABz sABz) (OH): NMR (300 MHz, d6-DMSO) δ11.22 (br,2H), 8.67 (s,1H), 8.65 (s,1H), 8.58 (br,2H), 8.54 (br,1H), 8.05 (d,4H,J=7 Hz), 7.5-7.7 (m,6H), 7.33 (m,2H), 7.2 (m,7H), 6.79 (dd,4H,J=7 Hz), 6.52 (t,1H,J=7 Hz), 6.47 (t,1H,J=7 Hz), 5.53 (d,1H,J=5 Hz), 5.37 (m,1H), 4.45 (m,1H) 4.34 (m,1H), 4.01 (m,1H), 3.69 (s,6H), 3.1-3.5 (m,5H(est.)), 2.7-2.95 (m,2H), 2.38 (m,1H).
Treated 21 mg of (DMTO) (ABz sABz) (OH) with 3 ml of saturated NH3/MeOH in a sealed vial for 3 days at RT. The solvent was evaporated and the residue was stirred with 1 ml of 80% HOAc/water at RT for 45 min. The solvent was removed using a vacuum pump/rotovap and a water bath at RT. Pure d(AsA) was obtained by HPLC: C8 Rainin 10�25 cm column (with guard), gradient=10-60% MeOH/water (linear), flow=3.5 ml/min, sample dissolved in 20% MeOH/water, 6c eluted at 21.2 min. NMR [partial spectrum] (300 MHz,d4-MeOH, all chemical shifts relative to MeOH @ 3.30) δ8.23 (s,1H), 8.20 (s,1H), 8.19 (s,1H), 8.11 (s,1H), 6.37 (m,2H), 5.22 (d,1H,J=6 Hz), 4.59 (m,1H), 4.33 (m,1H), 4.16 (dd, 1H,J=7,4 Hz), 3.74 (dq,2H,J=13,3 Hz), 3.47 (m,2H), 2.85-3.0 (m,2H), 2.69 (ddd,1H,J=13,6,1.5 Hz), 2.37 (ddd,1H,J=13,6,3 Hz).
To a solution of 200 mg of 5'-trityluridine (Sigma, St. Louis, Mo.) and 189 mg of 3'-O-t-butyldimethylsilyl-5'-sulfamoylazido-5'-deoxythymidine (from Example 4) in 2.5 ml of anhydrous acetonitrile under nitrogen was added 0.11 ml of triethylamine. After stirring at RT for 20 hr., the solvent was evaporated, and the residue was purified by chromatography on silica (1 to 7% methanol/DCM) giving 81 mg of the major product as one spot on TLC. Analytical HPLC shows a 50:50 mixture of products. Prep HPLC (C8 10�250 mm column with guard (Rainin), gradient: 80-90% over 25 min. of methanol/0.1M triethylammonium bicarbonate, flow=4 ml/min, sample: 10-15 mg/injection) afforded pure ((Ph) 3CO) [rU]sT(OTBDMS) (2' linked) and pure ((Ph)3CO)[rU]sT(OTBDMS) (3' linked). 2' linked: retention time=16.61 min.; NMR (300 MHz,d6-DMSO) δ11.47 (br,1H), 11.34 (br,1H), 8.39 (br,1H), 7.69 (d,1H,J=8 Hz), 7.25-7.4 (m,15H), 6.15 (dd,1H,J=7,6 Hz), 5.93 (d,1H,J=4 Hz ), 5.71 (m,1H), 5.49 (d,1H,J=8 Hz), 5.01 (m,1H), 4.42 (m,1H), 4.36 (m,1H), 3.99 (m,1H), 3.78 (m,1 H), 3.23 (m,2H), 2.25 (m,1H), 2.03 (m,1H), 1.77 (s,3H), 0.87 (s,9H), 0.09 (s,6H). 3' linked: ret. time=18.15 min.; NMR (300 MHz, d6-DMSO) δ11.46 (br,1H), 11.33 (br,1H), 8.37 (br,1H), 7.65 (d,1H,J=9 Hz), 7.35-7.5 (m,16 H), 6.13 (dd,1H,J=8,6 Hz), 6.07 (br,1H), 5.79 (d,1H,J=6 Hz), 4,87 (m,1H), 4.47 (m,1H), 4.34 (m,1H), 4.29 (m,1H), 3.80 (m,1H), 3.1-3.4 (m,7H), 2.24 (m,1H), 2.03 (m,1H), 1.76 (s,1H), 0.87 (s,9H), 0.08 (s,6H).
After dissolving 294 mg of (DMTO)ABz sT(OH) in 3 ml of dry DCM, 116 ul of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (Aldrich, Milwaukee, Wis.) and 31 mg of diisopropylammonium tetrazolide salt (prepared by mixing equal amounts of tetrazole and diisopropylamine) were added and the mixture was stirred for 1 hr. The reaction not complete by TLC (10% methanol/DCM). Another 60 ul of 4d and 15 mg of the tetrazole salt were added and stirring was continued for another 1 hr. The reaction was partitioned between half saturated bicarbonate and EtOAc. The organic layer was washed with brine, dried, and evap. The product was chrom. on silica (1-5% methanol/DCM with 0.5% pyridine) giving 452 mg of semi-pure (DMTO)ABz sT(OP(OCH2 CH2 CN)N(iPr2)). This material was dissolved in 3 ml of DCM and added this to 150 ml of pentane at -78� C. with stirring. The precipitate was filtered and collected. Repeated this procedure once. The white solid was evaporated from dry pyridine (2x) followed by dry toluene. Obtained (DMTO)ABz sT(OP(OCH2 CH2 CN)N(iPr2)) as white solid (used without furthur purification): 31 P NMR (300 MHz,d6-DMSO) δ151.133 (s), 150.596 (s) (minor impurities at 15-21 ppm); 1 H NMR (300 MHz,d6-DMSO) δ11.33 (br,1H), 11.21 (br,1H), 8.58 (s,2H), 8.52 (br,1H), 8.03 (m,2H), 7.1-7.7 (m), 6.80 (m,4H), 6.52 (t,1H,J=7 Hz), 6.15 (t,1H,J=7 Hz), 5.18 (m,1H), 4.46 (m,1H) 4.37 (m,1H), 3.9-4.1 (m), 2.88 (t,2H,J=6Hz), 2.77 (q,2H,J=6 Hz), 2.2 (m,1H), 1.76 (s,3H), 1.17 (m). mass spectrum (FAB) meas. 1161.76 (M+H), calcd. for C57H65N10O13PS+H 1161.43.
The oligodeoxynucleotide 5' d(GCGTGCATGC[AsT]CGTACG)3' (9c) was synthesized on the CODER automated DNA synthesizer on a 1 micromole scale using standard protocols with the block dimer phosphoramidite ((DMTO)ABz sT(OP(OCH2 CH2 CN)N(iPr2))) (from Example 10) in a 0.1M acetonitrile solution placed on the X port. The oligomer was cleaved from the solid support using 27% ammonium hydroxide with slow elution over 1 hr. This solution was put in a sealed vial and heated to 55� C. for 4 hr. After evaporation on a rotary evaporator, the crude product was purified by HPLC (C8 column, 10 mm�25 cm) using a linear gradient of 5-15% acetonitrile/0.1M triethylammonium acetate (TEAA). The oily product was evaporated from 50% ethanol/water until a white powder was obtained. The purified product had a retention time of 9.00 min. (C8 analytical 10 cm column, 1.0ml/min, 5-20% CH3CN/0.1M TEAA over 15 min.). A stock solution containing 5.0 ODs 9c/ml water (sterile) was prepared and stored in the freezer.
The buffer used was pH7.00, 10 mM aqueous sodium phosphate, containing 200 mM sodium chloride and 0.1 mM EDTA. The reference cells contained this buffer. Complementary strands (1.5 μM each) were allowed to anneal 15� C. and then the UV absorption at 260 nm was measured as the temperature was raised to 75� C. (0.4� C./min). The resulting melting curves are shown in FIG. 1. Solid diamonds show data from the oligonucleotide pair 9a:9c (estimated Tm =68� C.) and the open squares show data from the oligonucleotide pair 9b:9d (estimatedTm =72� C.). [See below for oligonucleotide sequences 9a, 9b, and 9d.]
The enzyme stock solutions used were 1.8 Units/ml of SVP, (phosphodiesterase I, Sigma, St. Louis, Mo.) and 147 Units/ml of AP (alkaline phosphatase, Sigma, St. Louis, Mo.). The buffer solutions used were tris (100 mM, pH 9) and MgCl2 (50 mM). The digestion was carried out in a 1.5 ml eppendorf tubes by incubating 0.1 Abs. units of the compound from example 14 with 25.6 ul of tris buffer, 24 ul of MgCl2 buffer, 10 ul of SVP stock solution, and 13.6 ul of AP stock solution at 37� for 18 hr. Next 10 ul of 2.5M sodium acetate was added followed by 250 ul of ethanol. After chilling to -78� for 30 min and centrifuging (12K rpm, 10 min), the supernatant was diluted to 1 ml with ethanol and respun. The supernatant was collected and evaporated on a speed-vac. The residue was redissolved in 1.0 ml of sterile water, and was stored at 4� until analyzed. FIG. 2a shows an HPLC chromatogram of a standard mixture of dC, T, dG, and dA. FIG. 2b shows an HPLC chromatogram of the product mixture resulting from prolonged digestion of oligodeoxynucleotide 9c. It can be seen that when the ODN containing the sulfamate ester linkage is digested for an extended length of time, the block dimer emerges intact. An HPLC chromatogram of a sample of the totally deprotected block dimer, d(AsT), which was independently synthesized (see example 19), confirmed that the peak labled as [AsT] had the same retention time as the synthetic material. This demonstrates that the sulfamate ester linkage is extremely resistant to exonucleases.
The oligonucleotide 9a was synthesized as described in Example 11 for 9c. Oligonucleotides 9b and 9d were synthesized by standard methods well known to those skilled in the art. The additional sequences are:
9a 5' d(CGTACGATGC[AsT]GCACGC)3'
9b 5' d(CGTACGATGCATGCACGC)3'
9d 5' d(GCGTGCATGCATCGTACG)3'
Each of the four oligomer strands 9a-9d (10-100 pmol ends) were 5'-end-labelled using T4 polynucleotide kinase (10 units) with γ-32 P ATP(3000 Ci/mmol). Strands were purified by phenol:chloroform extraction, chloroform extraction, ether extraction and two successive ethanol precipitations. Excess ATP was removed using a NACS PrePac column following the standard protocol (BRL, Bethesda, Md.).
The 5'-endlabelled single-stranded oligomers (12,000 cpm , ˜1 pmol) were annealed to their unlabelled complements (10 pmol) in 11 μl of buffer containing 50 mM tris-HCl (pH 8.0), 10 mM MgCl2 and 100 mM NaCl. The mixture was heated to 80� C. for 15 min and then cooled slowly to room temperature for 60 min. Restriction endonuclease Nsi I(10 units in 1 μl ) and 7 μl of enzyme reaction buffer was added to each of the annealed samples. The duplex DNA and Nsi I mixtures were placed at 37� C. for 60 min. The samples were ethanol precipitated with the addition of 10 μl of 7.5M ammonium acetate, 1 μl of tRNA and 90 μl of 100 % ethanol. Samples were centrifuged 15 min at 14,000 rpm at room temperature, supernatent liquid was removed and the DNA pellets were dried in vacuo. Samples were resuspended in 3 μl of formamide dye containing 80% deionized formamide, 0.1% xylene cyanol and 0.1% bromophenol blue, and were loaded onto a 20% denaturing polyacrylamide gel. Samples were electrophoresed for two hours at 2000 V. Gel was exposed to Kodak XAR film for 8-24 hours at -70� C. The results are shown in FIG. 3. Lanes 1 and 2 contain strand 9a annealed to strands 9c and 9d respectively; lanes 3 and 4 contain strand 9b annealed to strands 9c and 9d respectively; lanes 5 and 6 contain strand 9c annealed to strands 9b and 9a respectively; lanes 7 and 8 contain strand 9d annealed to strands 9b and 9a respectively. --S-- the sulfamate linkage and --P-- indicates the phosphodiester linkage.
The buffer used was 10 mM in magnesium chloride and Tris (base), and was adjusted to pH 7.5 with 0.1M HCl. The reference cell contained this buffer. The sample cell contained 0.660 OD units of poly U (Sigma, St. Louis, Mo.) and 0.330 OD units of d-AsA in 1.00 ml of buffer. The cells were equilibrated at 0.6� C. for 1 hour, and the temperature was ramped at 1� C./min. The melting profile is shown in FIG. 4. The Tm is estimated to be 15�-18� C. with a hypochromic effect of 35%. The reported literature value for the corresponding natural phosphodiester dimer, d-ApA, is 7.6� C.25. This example demonstrates that oligodeoxynucleotides containing only sulfamate linkage(s) can hybridize to their complementary unmodified counterparts and that the hybrid can have greater stability.
A solution of 1 mg of 3' linked ((Ph)3CO) [rU]-sT(OTBDMS) (from Example 9) in 3 ml of 27% ammonium hydroxide was incubated in a sealed vial at RT for 24 hr. The solvent was evap. When the residue was analysized by HPLC (C18 Rainin analytical column (4.6 mm�25 cm) with guard, flow of 1.5 ml/min, and a linear gradient of 80-90% methanol/water over 15 min.) none of the peak at 12.375 min. had converted to the 2'-linked ((Ph)3CO) [rU]sT(OTBDMS) (ret. time 11.110 min.). When a 1 mg sample of the 2' linked ((Ph)3CO) [rU]sT(OTBDMS) was treated in a similar manner, no equilibration was seen either. This demonstrates that sulfamate linked oligoribonucleotide derivatives are base stable with respect to cleavage and migration.
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31. Lamaitre, M., Bayard, B. and Leblew, B. (1987) Proc. Natl. Acad. Sci. USA, 84, 648-652.
32. "Oligodeoxynucleotides: Antisense Inhibitors of Gene Experssion", Topics in Molecular and Structural Biology series, CRC Press Inc., Boca Raton, Fla. (1989).
33. S. Benner, WO 89/12060.
34. P. S. Miller, J. Yano, E. Yano, C. Carroll, K. Joyaraman, and P. Tso, Biochem., 18, 5134-5143 (1979).
35. R. B. Waring, Nucl. Acids Res., 17, 10281-10293 (1989).
36. I. Yamamoto, M. Sekine, and T. Hata, J. Chem. Soc., Perkin Trans. 1, 306-310 (1980).
37. Charubala, Uhlmann, Beter, and Pfleiderer, Synthesis, 965 (1984).
38. T. -S. Lin, J. Pharm. Sci., 73, 1568-1570 (1984).
39. Oligonucleotide Synthesis: A Practical Approach, Ed. by M. J. Gait, IRL Press Ltd., Washington, D.C. (1984).
All references are incorporated herein.
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The Man in the High Castle
The Man in the High Castle is a science fiction alternate history novel by American writer Philip K. Dick. It won a Hugo Award in 1963 and has since been translated into many languages. The story of The Man in the High Castle, about daily life under totalitarian Fascist imperialism, occurs in 1962, fifteen years after the end of a longer Second World War. The victorious Axis Powers—Imperial Japan, Fascist Italy, and Nazi Germany—are conducting intrigues against each...... more on Wikipedia
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Tuesday, September 2, 2014
Edward Goodman, Salters - Funeral services for Edward Al Cat Daddy Goodman, conducted by the Rev. Olin Dukes, were held Saturday, August 23, 2014, at St. Mark Elementary School Gym of Kingstree, directed by Henryhand Funeral Home. Mr. Goodman died Saturday, August 16, 2014. He was born March 3, 1958, in Kingstree, the son of the late Rosa Mae and James Goodman. He was baptized at Laurel Swamp Baptist Church in Salters. He attended the public schools in Durham, North Carolina and Williamsburg County. He was employed by Duke University in Durham, North Carolina, Susan Florist of Kingstree, and the Mexican Restaurant of Kingstree. Four siblings, Rosa Lee Tisdale, Elma Goodman, Jimmy Lee Goodman, and Norris Goodman, preceded him in death. Survivors are: one daughter, Angela Goodman of Columbia; two sons, Quinton Goodman of Wake Forest, North Carolina; and Jimmie Goodman of Fayetteville, North Carolina; two sisters, Mary Shaw and Gertrude Brown; five brothers, James, Mason, Richard, Willis, and Calvin Goodman; and six grandchildren.
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text: A knit shirt is always a solid buy, something a little bit more up and up then a traditional t-shirt. The Brixton Hilt Knit Shirt is cool and collected. A durable, solid make, allover stripe pattern, and a left chest pocket with Brixton label detail. Constructed of 60% cotton, 40% polyester. Available in colorways as seen below. Model wears a medium.


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John Dillon. The new reality.
Others have warned Boeing that there will be a price to pay Washington workers who willingly bailed out manufacturing snafus in right-to-work and unskilled South Carolina and then received this corporate slap in the face. When we turn on each other, locals and nationals quickly separate in the clamoring for identity and fiscal security.
But it is Democrats in the New Reality that truly deserve another wary look by middle class workers, and for very good reason. What Fred Klonsky once described as a Bizarro World only endemic to Illinois has spread like an invasive species to many other places as well – take Washington State for example. In a recent Bloomberg article, Justin Bachman described important lessons to be taken from the Boeing Corp. win: Corporate threats now work. Getting the work back to Washington will make a better plane than the "good ole boys" of South Carolina. Boeing saves more, much more than the incentives offered to sign the contract. And…
Unions can no longer count on the Democrats.
Local Democrats in Washington State, like Iliinois Democrats, bent like reeds in the wind to the demands from above (Madigan or Quinn). In the case of Washington State, they "pleaded for workers to accept the Boeing contract and keep the company's workers in the region" (http://www.businessweek.com/articles/2014-01-09/boeings-victory-in-labor-fight-hurts-pensions-helps-the-777x). Likewise, in Illinois, Democrats are writing to their constituencies that they had to vote for SB1,"Even though it was 'the most difficult decision in my life.'"
Subtext: workers' unions in Illinois can no longer count on Democrats.
And some, like my Senator Jones (my Representative doesn't do communication) suggest that we can all revisit SB2404 when the Illinois Supreme Court finds SB1 unconstitutional.
Are you out of your ever-loving mind?
Read the entire post here.
David permalinkJanuary 11, 2014 8:51 pm
We need to give Dems a chance to live without unions this fall…………And of course Primaries. That is the only thing they understand now so that is what we must do. SB 2404 is in the trash bin Talking is over.
I would only add to this great post Boeing is one of the biggest Gov contractors. If we had any dems in DC they could have leaned on Boeing but they didn't …So don't rush to vote for them either…….
Does anyone know the nature of the pension that the Boeing workers had ? Did it have a COLA or was it more of an annuity ? That would mean those near retirement would not lose much because they could roll it into a private annuity if they want . The big effect will be on the new workers who will work for nothing
Anon permalinkJanuary 11, 2014 10:02 pm
In the 1930s the Democrats passed numerous laws to help and protect workers and unions. A lot of the Democrats today are DINOs, (Democrat In Name Only). The WAO unions negotiated in good faith and were stabbed in the back by the Democrats. The unions trusted them and now know better. If we win in court, that should be final, NO MORE OFFERS OF "COMPROMISE"!!!
Anonymous permalinkJanuary 12, 2014 9:23 am
I agree Anon, NO MORE OFFERS OF COMPROMISE….we already opened that box.
January 12, 2014 2:58 am
You all should also read about Boeing CEO McNerney (who is, BTW, a 1967 New Trier H.S. graduate {Winnetka resident} who then received his B.A. {don't see any further ed. on his Wikipedia profile} at Yale "where he excelled in baseball and hockey." Further information that one may Google involves Boeing's mandatory retirement age, which is 65. McN will turn 65 this August (it is expected that he will retire) and, to see HIS pension deal, go to http://www.bizjournal.com/boeing-ceo-mcnerneys-sweet-pension-deal.html. (I apologize if the link doesn't work, but anyone who's interested can easily find this information.) And–not to mention his current compensation has risen 15-20%–one source states $21.1 million while another is at $27.5 million.
BMMB permalinkJanuary 12, 2014 8:22 am
Green party, Labor party……. Ralph Nader has been saying it for years; this 2 party system has become a monopoly and it's time to end it. Let's vote for those 3rd party people and stop being afraid to do so. Also, I'm all for the 'none of the above' option on a ballot- I remember some guy saying "How humiliating and humbling would it be for someone to have to tell their children, 'I lost to no one' ". His point being he thought it would really raise the bar of candidates. I have to agree.
January 12, 2014 9:31 am
…What if this so-called "new reality" creates public school teachers who are willing to stop teaching in the public school classrooms across Illinois, and firemen who are willing to stop extinguishing fires in their towns and cities, and policemen who are willing to stop protecting peoples' homes and their communities, and other state employees who are willing to stop working for the municipal and state governments? Perhaps then the citizens of Illinois would recognize the value and service that public employees of Illinois provide; perhaps the citizens of Illinois would realize that the state employees' retirement plan is worth protecting and preserving just like the lives that these public employees have been safeguarding, supporting or assisting all along…
David permalinkJanuary 12, 2014 1:47 pm
Don't forget et the snowplow guys from last week ! What Glen describes is the US becomes the third world (see Kristoff todays NYT) I don't see any of the 1 percent rushing to live in any of those places Wonder how they will like it when……………..


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Posted on February 24, 2011 at 17:09 4 Comments 0 Likes
I created 2 groups for the State Parks and State of Arkansas just because.
You must be from Arkansas post your bird photos, tips, events,outings,
Soon I will have a page up on my website with all this information.
I wanted to get this going. Its from everybody IN Arkansas.
To help Arkansas State save our birds. I need 30 people to join to secure both sites for Arkansas State.
Please promote the links to Arkansas State and your friends.…Continue
Posted on February 21, 2011 at 16:46 0 Comments 0 Likes
© 2015 Created by Like2.com.


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Grant could lead to business expansion
The Rutherford County Commission and the Spindale Town Council jointly approved resolutions Friday to apply for grant through the North Carolina Rural Center to help with the potential expansion of a local business.
While the business was not named, the expansion calls for the creation of 53 new full-time jobs and a minimum investment of $530,000.
"The existing industries in Spindale and in Rutherford County continue to have opportunities to grow," said Matt Blackwell, Rutherford County Economic Development Commission director.
The total grant the county and Town of Spindale will be applying for totals $265,000 with a 5 percent match from the county and town. That match comes to a total of $13,250 with the county contributing $6,555 and the Town of Spindale putting in $7,376.
The county's match will come from the local Industrial Incentive Grant.
Blackwell said that the improvements to the un-named facility will equal the amount of the match required.
"We have estimated the improved value of the building and, over five years, we are looking at $13,250," Blackwell said.
Currently, the business employs 44 in a building with 135,000 square-feet. Blackwell said that the new jobs created, if the grant is approved, will have an average starting wage of $33,000 with benefits.
"The company also offers other fringe benefits along with that," Blackwell said.
Under the terms of the grant, the business will be expanding on their existing location and that expansion is eligible for reimbursement through the NC Rural Center Building Reuse Program for Occupied Buildings.
County Commissioners and the Spindale Town Council approved the resolutions during a public hearing on Friday at Spindale House.
Also, under the terms of the grant, the county nor the Town of Spindale were allowed to disclose the name of the business.


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Vinum Wine & Spirits Importing & Distributing
At Vinum, we are passionate about returning to the basics: about sharing good wines and spirits among family and friends. It is our mission to discover wines and spirits from artisan producers around the world and bring their drinks to your table. It's not about chasing wines and spirits you are told you must like; it's about helping you find variety and value, and reviving the excitement that comes from selecting and enjoying good wine and spirits.
Our goal is to import and distribute wines and spirits that:
- Represent outstanding value in the market place;
- We are truly passionate about;
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- Are from producers that have talent, and are great fun to work with.
Simply put, we want to bring you great wines and spirits that also represent great values! Our Portuguese, French and Spanish wines, as well as those from California, Washington and Oregon represent growers and winemakers who are at the top of their craft.
We hope that by exploring our wines and spirits you, too, will find they are worth being passionate about and that you will appreciate and enjoy them as much as we do.
We invite you to learn about our wines, wineries, spirits and distilleries highlighted on our website. If you have any questions about any of the wines, spirits or producers, or where you can buy any wines or spirits in our selection, please contact us.
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Pretty Little Liars
Shirtless Alert! Pretty Little Liars Star Tyler Blackburn Shows Off His Muscles (PHOTO)
It's always a happy day when Tyler Blackburn (Caleb Rivers) shows off his flat abs after taking a dip in the ocean. Admit it, your week just got a little brighter.
The Pretty Little Liars hunk was seen in Maui, Hawaii last week, spending time with a mystery blond girl at the Wailea Maui Marriott Resort and Spa. A few days later, Tyler was spotted sans blonde while swimming in the ocean, paddle-boarding, and jumping on a water trampoline. Is it just us, or does he look even sexier without a girl on his arm? Just saying!
Speaking of Tyler and the ladies, we're waiting to see what happens with his character and Hanna (Ashley Benson) on PLL now that they're broken up. Will the two get back together even though Hanna kissed Wren (Julian Morris) and Spencer's (Troian Bellisario) been flirting with Caleb? Time will tell!
Catch an all-new episode of PLL on Tuesday, August 7 at 8 p.m. ET/PT on ABC Family.
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When shopping for oneself or business, we tend to do what we can in order to find the best deal possible. Why? Inside we humans, there seems to be an ancient thread in the very fabric of our beings to hunt and gather what we need or want. Allow me to tug on that strand a bit this week as I show you some prime hunting grounds. Don your pith helmet and bring some extra socks (wet feet are never good) as we arm you to get the best bang for your buck!
Our first stop on this sales safari takes us to the concrete jungle of your local town. There the natives, sometimes known as "locals" or "glasswalkers," can best offer you advice and wares. Despite the size of your city, township, or village, there is a very good chance that anyone in business there has connections. In fact, you can even find special "chambers of commerce" or similar places which can help direct you to what you need.
Especially here in Sandusky, there are so many niche shops that the ability to find vendors, wholesalers, and retail opportunities is vast. Most of these "locals" are friendly and eager to help you, especially if you compliment them with a question. For instance, "That's a nice door chime you have, where did you buy it?" Or, "Wow, I can't believe your [local delicacy] tastes this good. Where did you learn to make this?" Don't be afraid to interact and talk with the merchants. I can personally attest to this method being extremely effective at producing leads on your hunt!
No matter how much you ask there are some times that a solution can't be found locally. So we need to hop back in our vehicle and enter the digital dimension of The Internet. It is here amid the mighty Google Mountains, the Bing Sea, and Prairies of Yahoo that we will explore. Often you can find through this medium a local source (many businesses keep online inventories), or at least one not too far away. But if exiting The Internet isn't possible for your interest or necessity, we'll go to a few established trading posts first.
Amazon.com is a name most of you are probably familiar with along with eBay and Half.com. These are great places to be able to find an array of products at comparable costs and shipping methods. Many shops also will freely ship your special order to a local branch that may not carry that particular product in stock. You wouldn't know from looking in-store, but our local Sam's Club can get you THIRTY-FIVE different styles of snack/drink vending machines for you, as one example. It never hurts to "browse global, shop local".
On the tech front, we will leave the waters of the Amazon and enter the beautiful bamboo glades of TigerDirect.com and Newegg.com. These two sites offer an amazing breadth of electronic product as well as consistent sales on everything from small parts to complete systems. If you don't have a local source of parts and service, try these (you can safely consider the refurbished items on each site too)!
Lastly, we will explore samples of the ever-shifting landscape of curiosity sites. These are places you can get odds and ends that you never thought existed, but may have a use for to own or sell. ThinkGeek.com is an amazing site with many nifty gadgets. You can spend a great amount of time there just musing over the geeky items, or wading through still pools of nostalgia. Accoutrements.com is well worth exploring too. Woot.com is a "daily deal" site that features one item per day on sale at an incredible price. I bought two new security camera systems there rather inexpensively! Several kinds of "woots" are available every day from shirts to wine to tech. Groupon.com is similar and has witty and descriptive writing just like Woot.
It has been another fond journey with you, friend reader, so I wish you good luck and happy hunting for yourselves or your business! If you need help finding anything please feel free to ask and I'll do my best to guide you to the right destination. Au revoir!


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- Infowars - http://www.infowars.com -
Millions falling into poverty in recession-racked Italy: report
Posted By Adan On May 23, 2013 @ 12:06 pm In Economic Crisis,Old Infowars Posts Style | Comments Disabled
May 23, 2013
Millions of Italians cannot afford to heat their homes properly or eat meat as their country is racked by recession and soaring unemployment, said a report which found the number of people considered seriously deprived had doubled in the past two years.
The findings from national statistics institute ISTAT underline the scale of the challenge faced by the new coalition government of Enrico Letta, which has vowed to stimulate growth and tackle a youth jobless rate of almost 40 percent.
A recession that has lasted almost two years has taken a heavy toll on ordinary Italians who are increasingly digging into their savings, ISTAT said in its annual report.
Article printed from Infowars: http://www.infowars.com
URL to article: http://www.infowars.com/millions-falling-into-poverty-in-recession-racked-italy-report/
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Universal battery charger recharges all batteries, including alkaline, Ni Cad, high power and zinc carbon; extends battery life up to 10 times! Saves you money and reduces environmental waste, too. Six charging bays hold AA, AAA, C, D, and 9 volt sizes. LED monitor indicates battery status and charging levels at a glance. Includes AC adapter. 11½" x 7½" x 2½".
Welcome to Harriet Carter - Distinctive Gifts Since 1958. Find a large selection of products for organizing your life, as well as As Seen On TV products, household gadgets and problem solvers, unique gifts and gift ideas. Buying online is the fastest and most convenient way to shop! We have created this website just for you; everything is arranged by product category for quick, easy selection. In addition to our selection of gift ideas, you'll find products for organizing, As Seen On TV items, New Arrivals, Web Exclusive Gifts, Household Gadgets and products for your Pets.
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We are now accepting PayPal as a form of payment. For those of you unfamiliar with PayPal, it is a Web-based service that enables you to pay an online merchant without revealing your financial information. We've added this payment option to better meet your expressed needs for confidence, control, convenience and choice in your payment methods.
Learn more about PayPal and decide if you would like to set up a PayPal account.
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ESP provides powerful and effective leisure management software solutions to many of Europe's leading Organisations and Clubs. With over 20 years at the forefront of the industry, we have the knowledge and experience to deliver tailor-made systems that are easy to use and represent excellent value for money. We are the preferred partner for many Local Authority Sports Facilities, Members Clubs, Universities, Leisure Trusts, Spas, Hotels and Golf Clubs.
We offer a comprehensive and scalable portfolio of software modules that work stand-alone or combined to suit all sizes of operation, backed by our friendly 24/7 support service. With unrivalled functionality and intuitive interface we are the one-stop solution for membership management, CRM, bookings, retail, food & beverage, internet services, hotel, reporting and much more.
11th March
The ESP team will be heading up to the Marriot Forest of Arden Resort on the 16th and 17th of March for the 2015 GolfBic Conference.
ESP services and solutions are designed to give your business the flexibility and control you need and the club 'experience' your customers want. We are really looking forward to showcasing our latest product and services innovations and we will be demonstrating all of our new mobile and tablet technologies at GolfBic 2015.
Come and see the team, we have a lot to chat with you about!


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Given her beauty she could sell ice to Eskimos, and Bar Refaeli was busy with a new ad campaign for Buick this week.
The 30-year-old Israeli supermodel worked with celebrity yoga instructor Chad Dennis and held a few poses to show how Buick vehicles actually integrate yoga and driving.
It's getting closer and closer to the release of "Maze Runner: The Scorch Trials" and 20th Century Fox just unveiled a new trailer for all to enjoy.
The forthcoming sequel stars Dylan O'brien, Thomas Brodie-Sangster, Kaya Scodelario, Will Poulter and Patricia Clarkson and is slated to hit theaters on September 18th.
It's been a long and challenging road through her high school years and finally Kylie Jenner has received her diploma!
On Thursday (July 23) the "Keeping Up with the Kardashians" star enjoyed a surprise graduation party, organized by her mom Kris Jenner and hosted by "American Idol" hunk Ryan Seacrest.
Kicking off the weekend a little bit early, Nicole Scherzinger got all done up in a backless snakeskin dress and hit up the swanky Capsule Collection Launch from Michael Costello and Style PR last night (July 23).
Her new movie "Trainwreck" is full of laughs and love, and Amy Schumer is distraught that a gunman opened fire in a Louisiana movie theater killing two moviegoers and injuring nine additional people.
According to multiple reports, a 58-year-old white man was at a screening of "Trainwreck" in Lafayette, Louisiana when he pulled a gun and tried to flee the theater along with the audience members who were running out the door.
It was only a matter of time before her kiddos started getting into showbiz and Angelina Jolie is slated to work with her son Maddox Jolie-Pitt on a new Netflix Original Film.
The "Mr. and Mrs. Smith" starlet will direct an adaptation of "First They Killed My Father: A Daughter of Cambodia Remembers," a novel written by Loung Ung. Maddox is originally from Cambodia and will be "involved in the production of the film."
Taking care of a longstanding issue, Sean Diddy Combs had knee surgery on Thursday (July 23) and it sounds like it was pretty painful.
After he was discharged from the hospital, the "Bad Boy For Life" rapper posted a photograph of himself via Instagram, sitting in a wheelchair and holding a pair of crutches.
Showcasing her sexy form, Martha Hunt wears a series of bathing suits while posing up a storm in the latest issue of Hamptons magazine.
And while many of her peers are all about being thin, the 26-year-old Victoria's Secret Angel insists she will never be irresponsible with her image.
"Krissy was and is an angel. I am completely numb at this time. My family must find a way to live with her in spirit and honor her memory. Our loss is unimaginable. We thank everyone for the prayers for Krissy. And our family as we mourn my baby girl."


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Pretty Little Liars
Is Showrunner Marlene King Going to Leave Pretty Little Liars for Her New Show, Now & Then?
Pretty Little Liars showrunner Marlene King is currently working on developing a new project for ABC Family, a TV version of the classic '90s coming-of-age flick Now & Then, which she wrote. This is exciting news, but it leaves some fans worried that Marlene might jump ship from PLL to the new project.
But never fear, PLL fans: Marlene plans to stay faithful to her first show.
A fan recently asked Marlene via Twitter, "Can you work both PLL and The Now & Then series at the same time?"
Marlene replied, "that's the plAn."
Heading up two shows might seem like a lot, but Marlene would be far from the first showrunner to tackle that task. For example, TV bigwig Shonda Rhimes is currently the showrunner on not two but three shows: Grey's Anatomy, Private Practice, and Scandal. If Shonda can do it, so can Marlene!
Catch an all-new episode of PLL on Tuesday, July 24 at 8 p.m. ET/PT on ABC Family..
Source: Twitter, Twitter
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