Phosphorothioates, chiral

Chirahty at the phosphoms is an unavoidable problem in all phosphorothioate syntheses. The phosphoramidite method produces a mixture of both the and the diastereomers having a small excess of the isomer (53). Although some progress has been made in the chiral synthesis of dinucleoside phosphorothioates, low yields have limited the utility of these approaches. The chiral center may be eliminated by replacing the other, nonbridging oxygen with sulfur. Avoidance of the chirahty problem is one reason for the interest in phosphorodithioates. 

Example 3 different activators derived from 4,5-dicyanoimidazole were prepared and used by Just and Lu in the stereoselective coupling reactions of phosphoroamidites with nucleosides [19]. An easily removable chiral auxiliary derived from d-xylose was synthesized and used for stereoselective construction of dithymidine phosphorothioate. The part of this study is illustrated below. 

The stereochemical mechanism of 3 processing and strand transfer has been investigated using DNA substrates that incorporate phosphorothioate link-ages [8]. For both reactions, the introduced chiral centers are inverted in the products, implying that the reactions occur via a one-step in-line displacement mechanism rather than via a covalent intermediate. 

Notice that the negative charge is largely localized on sulfur in these phosphorothioate compounds.696 More general is the use of 170 and lsO to form a chiral phospho group ... 

Stereochemical studies support in-line mechanisms for both the transesterification and hydrolysis steps of ribonuclease catalysis. For example, chiral uridine 2, 3 -cyclic phosphorothioates are hydrolyzed with inversion of configuration, with the diastereoisomer shown yielding a 2 -monophosphothioate of the R configuration at phosphorus. 

R0P032 is pra-prochiral, since two oxygen atoms must be substituted to produce a chiral compound. Chiral phosphates have been synthesized de novo by using stereospecific chemical and enzymatic reactions with isotopic and/or atomic substitutions. For example, a chiral phosphorothioate may be synthesized from a prochiral phosphate by replacing an oxygen atom with a sulfur atom. Similarly, what would otherwise be a pro-prochiral phosphate has been synthesized as a chiral product by replacing one oxygen atom with sulfur and another... 

With regard to the phosphorus stereochemistry, a new stereospecific synthesis of chiral 0,0-dialkyl thiophosphoric acids (6,7) has been developed which is based on the Horner-Wittig reaction of the optically active phosphonothionate carbanions containing the sulfoxide or dithioacetal moieties. The transformation of (R)-(+) 0-methyl 0-isopropyl methanephosphonothionate (10)(8) into (R)-(-) 0-isopropyl phosphorothioic acid (H) best Illustrates this method. 

Since it is well established that the P-chiral phosphorothio-ates serve as effective probes in mechanistic studies for the phos-phoryl group transfer enzymes /16/, we turned our attention to the application of diastereomeric 8 (B=Thy, Ar=pN02C6Hi -,/17f) to elucidate the mode of action of spleen phosphodiesterase (SPDE, EC 3.1.1. 18). This enzyme splits the phosphodiester bonds to yield nucleoside 3 -phosphates. In the case of it was expected that its SPDE-catalyzed hydrolysis in 180 H 2O medium leads to P-chiral thymidine 3 - 180 phosphorothioate. On the contrary to our expectation the main product of this reaction was thymidine cyclic 3 ,5 - Rp phosphorothioate (10) /6/. By treatment of 8 under the same conditions, but in the absence of the enzyme, no trace of J 0 was detected. ... 

ATPases. A few pyrophosphotransferases catalyze substitution at Pg. P0 and Pg are prochiral centers and can be made chiral by stereospecific replacement of one or the other diastereotopic oxygen with sulfur or enrichment with lsO or nO. Py is an achiral center which can be made chiral either by replacement of one of the three equivalent oxygens with sulfur and stereospecific enrichment of another with 180 to give a chiral [180]phosphorothioate, or by stereospecific enrichment of one with nO and another with lsO to give a chiral [160, nO, lsO]phosphate. 

The second prerequisite for a stereochemical investigation is that an effective method for assigning configurations at the chiral phosphorus centers in substrates and products must be available. The methods in current use include X-ray diffraction crystallography, mass spectrometry, nuclear magnetic resonance and circular dichroism. The methods by which chiral phosphorothioates can be synthesized and configurationally analyzed are discussed in this section. 

The first chiral phosphates to be used for stereochemical analyses were chiral phosphorothioates, which were used to determine the stereochemical courses of ribonuclease, UDP-glucose pyrophosphorylase, adenylate kinase and several other kinases and synthetases. The chiral phosphorothioates either had sulfur in place of an oxygen at an otherwise prochiral center of a phosphodiester or phosphoanhydride or stereospecifically placed sulfur and 180 (or nO) in a terminal phosphoryl group. The syntheses and configurational analyses of the most important of these compounds are outlined in the following. 

The chiral y-[l80]phosphorothioate of ATP, (Pp)-ATPyS, yl80(/3, y)lsO, was synthesized by the procedure outlined in Fig. 8 [25]. (Sp)-ADPaS, alsO(a,/3)l80 was prepared by stereospecific phosphorylation of AMPaS, al802 using the adenylate and pyruvate kinase system followed by dephosphorylation with glucose and hexokinase. This was the starting material for the synthesis, with the chiral a-... 

There are two basic methods by which configurations are assigned to chiral [lsO]thiophosphoryl groups. The first one is the one introduced by Usher, Richardson and Eckstein [31], which is applicable to vicinal hydroxy [180]phosphorothioates when the absolute configurations of the corresponding diastereomeric cyclic phos-phorothioates are known. The principle is illustrated in Equation 8 for sn-glycerol-3-[18 OJphosphorothioate ... 

Fig. 14. Orientations of enzymatic phosphorylations of chiral [lsO]phosphorothioates.. Shown are the orientations of phosphorylation of AMPS by adenylate kinase and ATP and the phosphorylation of ADP/3S by pyruvate kinase and phosphoenolpyruvatc (PEP) and by acetate kinase and acetvl P...

Fig. 15. Configurational analysis of chiral [ lsO]phosphorothioates by stereospecific phosphorylation.

Despite the broad utility of chiral phosphorothioates, certain enzymes, such as alkaline phosphatase and phosphoglycerate mutase, do not accept phosphorothioates as substrates. Stereochemical studies of these enzymes awaited the development of methods to synthesize and assign configurations to chiral phosphates. Chiral [l60, l70,180]phosphomonoesters and [l8OJphosphodiesters have been elegantly... 

Tsai and Chang showed that 5 -nucleotidase-catalyzed hydrolysis of AMPS to adenosine and phosphorothioate proceeds with overall inversion of configuration [28], They synthesized the (Rp) and (Sp) isomers of AMPS, l80 used them as substrates for 5 -nucleotidase in H(70, and determined the configurations of the two samples of [ l60, nO, 180]phosphorothioate by the procedure of Webb and Trentham outlined in Fig. 18 [48]. The configurations of chiral phosphorothioate samples obtained from the (Rp) and (Sp) isomers of AMPS, l80 were (/ p) and (Sp), respectively, the configurations corresponding to inversion. The stereochemistry was thereby shown to be that of Equation 13 ... 

A claim that the stereochemical course of cyclic AMP phosphodiesterase action on 3, 5 -cyclic AMPS and 3, 5 -cyclic AMP, l80 differed, inversion for the chiral cyclic phosphorothioate and retention for the chiral cyclic phosphate, resulted from... 

A difficulty is that the phosphorus centers in DNA are not chiral, because two of the groups bound to the phosphorus atom are simple oxygen atoms, identical with each other. This difficulty can be circumvented by preparing DNA molecules that contain chiral phosphoryl groups, made by replacing one oxygen atom with sulfur (called a phosphorothioate). Let us consider EcoKV endonuclease. This enzyme cleaves the phosphodiester bond between the T... 

Table 2 lists phosphatases for which the stereochemical outcome has been determined by using chiral phosphomonoester substrates. In such experiments the phosphoryl group is made chiral using lfiO, 170, and lsO, or, a phosphorothioate substrate is used that has a sulfur atom and two isotopes of oxygen in the nonbridging positions. Summaries of results for additional enzymes that catalyze phosphoryl transfer are available in other reviews.20,76,77... 

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