Beyond Triphosphates: Reagents and Methods for Chemical Oligophosphorylation

Abstract

Oligophosphates play essential roles in biochemistry, and considerable research has been directed towards the synthesis of both naturally occurring oligophosphates and their synthetic analogs. Greater attention has been given to mono-, di-, and triphosphates, as these are present in higher concentrations biologically and easier to synthesize. However, extended oligophosphates have potent biochemical roles, ranging from blood coagulation to HIV drug resistance. Sporadic reports have slowly built a niche body of literature related to the synthesis and study of extended oligophosphates, but newfound interests and developments have the potential to rapidly expand this field. Here we report on current methods to synthesize oligophosphates longer than triphosphates and comment on the most important future directions for this area of research. The state of the art has provided fairly robust methods for synthesizing nucleoside 5’-tetra- and pentaphosphates as well as dinucleoside 5’,5’-oligophosphates. Future research should endeavor to push such syntheses to longer oligophosphates while developing synthetic methodologies for rarer morphologies such as 3’-nucleoside oligophosphates, polyphosphates, and phosphonate/thiophosphate analogs of these species.

Graphical Abstract

Introduction

Chemical synthesis and the study of biological systems have naturally progressed in tandem, with synthesized biomolecules and their non-natural analogs playing a key role in many biochemical experiments.¹ Accordingly, whole fields of synthetic chemistry have arisen around the synthesis of preeminent classes of biomolecules such as peptides,^2–4 oligonucleotides,^5–7 carbohydrates,^8,9 etc. Phosphorus, as an essential element for all life,¹⁰ is necessarily included in these synthetic pursuits, especially in the study of nucleotides, DNA/RNA, and protein phosphorylation. However, in contrast to the rich field of carbon bond centric organic chemistry, biorelevant phosphorus chemistry is less well developed. Phosphorus exists in biological systems almost exclusively as P(V) phosphates,¹¹ and phosphate synthesis is mostly limited to simple nucleophilic substitution reactions, despite the frequent departure from biological conditions to utilize P(III) chemistry. Typically, phosphorus bound leaving groups are substituted with a nucleophilic substrate such as an amine, alcohol, or phosphate of interest (Figure 1, Top). This general methodology is simple but powerful enough to achieve transformations as difficult as oligonucleotide synthesis.⁵ However, one of the most important properties of phosphates is their ability to form longer linear or cyclic oligophosphates,¹² such as adenosine triphosphate (ATP) and other nucleoside 5’-triphosphates (NTPs). The synthesis of oligophosphates has remained an enduring challenge, and selectively synthesizing extended oligophosphates requires pushing the limits of phosphorylation chemistry.

Figure 1.

Top: A generalized phosphorylation reaction in which a nucleophile substitutes a leaving group from an electrophilic phosphate. Bottom: Examples of extended oligophosphates present in biological systems: nucleoside 5’-oligophosphates (p_nN), dinucleoside 5’,5’-oligophosphates (Np_nN), and inorganic polyphosphate (PolyP).

ATP is perhaps the most important and well known biological cofactor, acting as the primary mediator of energy transfer in cellular processes.¹³ ATP and the other NTPs are also the monomers for biological DNA/RNA synthesis¹⁴ as well as signaling molecules,¹⁵ resulting in paramount utility of these compounds and their analogs for biochemical studies and drug candidates.^16,17 Accordingly, the chemical synthesis of NTPs has received considerable attention, beginning with the first synthesis of ATP by Khorana in 1954 through N,N’-dicyclohexylcarbodiimide (DCC) mediated coupling of adenosine monophosphate (AMP) and orthophosphate.¹⁸ Since then, considerable research effort has been devoted to developing improved syntheses of NTPs, resulting in an at times confusing variety of reaction conditions and methods.^17,19 However, NTPs are far from the only oligophosphates utilized by Nature. Nucleoside 5’ tetra- and pentaphosphates (Np₄ and Np₅)^20–22 have been identified in biological sources as well as more complex morphologies such as dinucleoside 5’,5’-oligophosphates (Np_nNs)^23,24 (Figure 1, Bottom). Some of these compounds and their analogs have been known for decades and even investigated as therapeutics in clinical trials,²⁵ but the difficulty of isolating such low abundance compounds from natural sources and dearth of synthetic methodology has hampered their study. Phosphates are also found in biology in the form of long inorganic polyphosphates (PolyP, up to 100s of phosphate units long)²⁶ and more recently, as oligo-²⁷ and polyphosphorylated peptides²⁸ (Figure 1, Bottom). While many lessons and methodologies from NTP synthesis can be applied to extended oligophosphates, research on the synthesis of these molecules is much rarer.

In this Perspective, we will review the established and cutting edge methods for chemical oligophosphorylation with an emphasis on the synthesis of oligophosphates longer than triphosphates. As an oligophosphorylation reagent or method can be utilized to generate products with a variety of different phosphate chain lengths, this Perspective is structured into sections highlighting methodologies for appending different numbers of phosphate units, that is monophosphorylation, diphosphorylation, etc. Although not the prime focus of this Perspective, the applications, biological and otherwise, of extended oligophosphates of defined length, as well as PolyP that contains a distribution of chain lengths, will be discussed briefly.

General Considerations and Purification

Phosphates, as oxyanions, are encountered either as salts, neutral acids, or a combination thereof with major consequences for their reactivity. In aqueous systems, these salts are commonly those of alkali metal or ammonium (NH₄) cations. However, many of the activating agents used in synthetic phosphorylation chemistry are either water reactive or insoluble in water. Furthermore, electrophilic phosphorus species are often unstable with respect to water as well. Therefore, phosphorylation reactions conducted in water are usually limited to enzymatic reactions,^29,30 certain “green” reactions,³¹ some reactions of phosphorimidazolides,^32,33 and those using specialized water soluble reagents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC).³⁴ Synthetic phosphorylation chemistry is typically conducted in polar organic solvents such as acetonitrile, dimethylformamide (DMF), and pyridine, requiring the use of lipophilic cations. For these transformations, phosphates are often encountered as tetrabutylammonium (TBA) or less soluble tributylammonium salts. These lipophilic salts are readily accessed by first acidifying the desired phosphate compound with an acidic resin, such as Dowex 50WX8, and then neutralization with either TBA hydroxide or tributylamine, followed by lyophilization.³⁵ The bis(triphenylphosphine)iminium (PPN) cation has also been used and typically gives more crystalline phosphate salts.³⁶ The nucleophilicity of phosphates is enhanced when they are deprotonated, and nucleotide substrates are commonly encountered fully deprotonated, such as the salt [TBA]₂[AMP].³⁵ However, especially in the absence of hydrogen bond donor groups, retention of some protons is necessary for stability in organic solvents. For example, [TBA]₃[HP₂O₇] is readily prepared and soluble in polar organic solvents, but [TBA]₄[P₂O₇] is accessible only in water.

Most synthetic phosphorylation chemistry can be divided into reactions that utilize P(III) reagents and those that operate in an entirely P(V) regime. Prime motivations for P(III) chemistry are the greater reactivity of these reagents and facile access to thiophosphate products. However, in addition to requiring air free conditions, the great reactivity of P(III) reagents often, although not always,^37,38 necessitates the use of protecting groups, for example of nucleoside 2’ and 3’ positions. In a typical example, an electrophilic P(III) compound bearing a leaving group is synthesized, ultimately from phosphorus trichloride. Treatment with a nucleophile, followed by oxidation or sulfurization yields the desired phosphate or thiophosphate product (Figure 2, top). P(V) methods usually start with a phosphate of interest that is then activated to form an electrophilic compound. Common activating agents are sulfonyl chlorides,³⁵ carbodiimides,¹⁸ carboxylic acid anhydrides,³⁹ and carbonyldiimidazole (CDI).⁴⁰ Importantly, phosphates contain multiple nucleophilic oxygen positions, and treating an inorganic phosphate with an activating agent results in oligomerization, especially cyclic metaphosphates.^41,42 Similarly, treatment of a substituted triphosphate with an activating agent results in an intramolecular dehydration to form a substituted trimetaphosphate (TriMP) compound (Figure 2, bottom). However, the resulting metaphosphate is still a powerful electrophile due to the phosphotriester functionality, and ring opening of these intermediates has been used repeatedly in the synthesis of oligophosphates.^{35,37,38,43–46}

Figure 2.

Top: General P(III) phosphorylation conditions in which a P(III) electrophile is treated with a nucleophile followed by oxidation or sulfurization. Bottom: General methods for electrophilic activation of P(V) phosphates in which a mono- or diphosphate is treated with an activating agent to generate a leaving group, or an extended oligophosphate is treated with an activating agent to generate an electrophilic substituted metaphosphate.

The purification of oligophosphates is often the most difficult and expensive part of a synthetic procedure. As mixtures of phosphate salts with similar solubilities, purification of crude oligophosphates by crystallization alone is extremely rare. The standard purification method is chromatography, typically High Performance Liquid Chromatography (HPLC) for small scales and flash or medium pressure Liquid Chromatography (LC) for larger scales. Traditional reverse phase (RP) chromatography is a poor separation method for most biological oligophosphates, as these highly polar compounds elute rapidly. In contrast, anion exchange (AX) chromatography strongly retains anionic oligophosphates and is the most common purification method.⁴⁷ Ion pairing RP-HPLC⁴⁸ and hydrophilic interaction chromatography (HILIC)⁴⁹ methods are also sometimes used for purification. Electrophoresis methods, such as Capillary Electrophoresis (CE)^50,51 and Polyacrylamide Gel Electrophoresis (PAGE),⁵² can also show good efficiency for oligophosphate separation, but they are not typically applied on preparative scales. A key target in continuing phosphorylation chemistry is the development of high yielding reactions and other methods to avoid or simplify costly chromatographic separations.

Mono-, Di-, and Triphosphates

The many diverse biological roles of monophosphates, diphosphates, and triphosphates is a vast topic that is intimately intertwined with all aspects of biochemistry. Extremely briefly, such phosphates mediate energy transfer, enzyme regulation, DNA/RNA synthesis, signal transduction, bone growth, and much more far beyond the scope of this Perspective.⁵³ Nevertheless, it behooves us to examine the general synthetic methodologies used to access these species and how these relate to the synthesis of extended oligophosphates. The synthesis of nucleoside mono-, di-, and triphosphates has also been reviewed several times.^17,19,54

Synthesis of monophosphates is limited to coupling a desired organic substrate (such as a nucleoside) and a monophosphate synthon (either P(III) or P(V)). In a simple example, the Yoshikawa method, nucleosides are treated with phosphoryl chloride in alkylphosphate solvents to selectively phosphorylate the 5’ position (Figure 3).⁵⁵ In a much more complex example, P(III) chemistry is used in the phosphoramidite method for the synthesis of oligonucleotides (Figure 3).⁷ In this method, the 5’-hydroxyl position of a silica supported oligonucleotide is coupled with a P(III) 3’-phosphoramidite nucleoside bearing a 4,4’-dimethoxytrityl (DMT) protecting group on the 5’ position and a 2-cyanoethyl protecting group on the phosphoramidite oxygen. After coupling, the product is capped with acetic anhydride and a nucleophilic base to prevent chain elongation of any remaining unreacted 5’ hydroxyl groups. The phosphorus center is then oxidized, and the DMT group is cleaved with acid, providing a free 5’ hydroxyl group for further oligonucleotide chain extension. After the full oligonucleotide is synthesized, basic conditions cleave the molecule from the silica support and deprotect the 2-cyanoethyl groups. A related method of oligonucleotide synthesis that allows incorporation of thiophosphate moieties has also been introduced recently by Baran.^56,57

Figure 3.

Representative syntheses of mono-^7,55 and diphosphates.^40,58

In principle, the synthesis of diphosphates can be performed either by coupling an organic substrate and a diphosphate or by monophosphorylation of a monophosphate substrate. The former is analogous to monophosphorylation, but diphosphate electrophiles are rare. An example of such a species is a mixed P(III)-P(V) methylene bridged analog of pyrophosphate used by Filipov⁶³ and Fiedler⁶⁴ to synthesize bisphosphonates. In an unusual phosphorylation example, Poulter used pyrophosphate as a nucleophile to react with an electrophilic organic substrate, the opposite philicities of most phosphorylation chemistry (Figure 3).⁵⁸ In contrast to scarce diphosphorylation chemistry, utilizing a monophosphorylation procedure with a monophosphate substrate is typically quite facile, due to the great nucleophilicity of monophosphates. For example, NMPs react with CDI to form electrophilic phosphorimidazolides. Subsequent reaction with another monophosphate substrate results in a diphosphate (Pankiewicz, Figure 3).⁴⁰ Phosphorimidazolides have been used extensively for such chemistry,¹⁷ with notable improvements such as aqueous conditions and faster reactions due to added zinc salts.³²

Similarly, the synthesis of triphosphates can be performed either by (1) coupling an organic substrate with a triphosphate source, (2) diphosphorylation of a monophosphate, or (3) monophosphorylation of a diphosphate. The first method is the most recent development, pioneered by Taylor,^{35,59,61,65,66} in which a nucleophilic substrate is treated with a leaving group modified trimetaphosphate (Figure 4). Notably, Taylor’s initial methodology relies on in situ activation of TriMP, but subsequent reports have described isolation and structural characterization of analogous activated TriMP species as crystalline compounds (Figure 4).^61,62 Jessen^37,38 and Huang⁶⁷ have elaborated similar triphosphorylation approaches that instead utilize mixed P(III)-P(V) analogs of TriMP. The second route to triphosphates is the most classic, including Khorana’s original ATP synthesis,¹⁸ and the well known Ludwig-Eckstein synthesis of NTPs (Figure 4).⁶⁰ These methods have significant drawbacks in terms of yield and purification, and Chaput has addressed these issues by utilizing a pyrene modified pyrophosphate reagent.⁶⁸ In addition to a more efficient coupling step, the pyrene modification allows purification by normal phase chromatography, significantly improving the cost and scalability of purification. Lastly, the third approach is analogous to the monophosphorylation procedures previously discussed.^69,70 In this manner, iterative monophosphorylation reactions can be used to convert monophosphates into diphosphates and then triphosphates, such as the work by Jessen^71,72 and Chaput’s synthesis of TNA triphosphates.⁷³ However, diphosphate substrates are much less nucleophilic than monophosphate substrates, often resulting in longer reaction times and lower yields.³⁷

Figure 4.

Representative syntheses of triphosphates.^18,59,60 Thermal ellipsoid plots of crystallographically characterized triphosphorylation reagents^61,62 ([PPN][P₃O₉P(NC₄H₈)₃]·2H₂O, [PPN][HNEt₃][P₃O₉Ts]) with thermal ellipsoids set at 50% probability and hydrogen atoms, cations, and solvent molecules omitted for clarity (red for oxygen, orange for phosphorus, blue for nitrogen, grey for carbon, yellow for sulfur). The thermal ellipsoid plot of [P₃O₉Ts]²⁻ was adapted from reference 62 as permitted under its CC BY 4.0 License https://creativecommons.org/licenses/by/4.0/.

Biological Synthesis of Oligophosphates

Although there are notable examples of enzymatic syntheses of oligophosphates,^17,74,75 the nonaqueous synthetic chemistry focused on in this Perspective is a large departure from native biological syntheses of oligophosphates. However, the biological synthesis and utilization of these compounds is both a source of inspiration and a vital biochemical application for synthetic oligophosphates. Phosphorylation and dephosphorylation enzymes are broadly divided into phosphotransferases, phosphorylases, and phosphatases, representing an extremely broad range of enzymes.⁷⁶ Kinases are perhaps the most familiar phosphotransferase subclass, catalyzing phosphoryl transfer from ATP to form phosphorylated substrates and ADP (Figure 5). This is analogous to synthetic monophosphorylation reactions, with ADP acting as a leaving group (Figure 5). The vast number (~500 known human kinases)⁷⁷ and high selectivity of kinases indicate the need for careful preassociation of substrates and hydrogen bonding networks that enzyme active sites provide. Overall, these aqueous phosphorylation conditions are quite mild compared to the water reactive and highly electrophilic phosphorus reagents that are common in synthetic phosphorylation chemistry. Additionally, ATP, the most common source of biological phosphoryl equivalents, is almost entirely bound to magnesium under physiological conditions. This divalent cation is important for screening charge and facilitating phosphorylation reactions,⁷⁸ a strategy that has been widely applied to difficult synthetic phosphorylations.^{32,35,37,46,79}

Figure 5.

Generalized reaction scheme of a kinase.

Although nucleotides are a stereotypical class of oligophosphates, biology also extensively utilizes inorganic polyP.^26,80 These compounds have been known for decades across all kingdoms of life, particularly due to the presence of polyP granules in bacteria that are visible through microscopy. In bacteria, polyP is synthesized and utilized by polyphosphate kinases (PPK1 and PPK2) which transfer the terminal phosphate from ATP to polyP as well as the reverse reaction.⁸¹ A recent solid state NMR study backs up previous tentative reports of cyclic metaphosphates in these polyphosphate granules,⁸² but the potential biological importance of metaphosphates is an open question. PolyP is present in mammals as well and is involved in blood clotting.^83,84 The biosynthesis of polyP in humans has been an enduring mystery.⁸⁵ A recent report detailed synthesis of PolyP by F₀F₁-ATP synthase in mammalian mitochondria,⁸⁶ and this result awaits further validation as the source of human polyP.

A few groups have developed preparative enzymatic syntheses of extended oligophosphates. As early as 1989, Tomita utilized Leucyl t-RNA synthetase for the synthesis of adenosine 5’-tetraphosphate (p₄A) and diadenosine 5’,5’-tri-, tetra-, and pentaphosphates (Ap₃A, Ap₄A, Ap₅A).⁸⁷ The authors proposed that the enzyme reacts with ATP to form an activated AMP complex. Subsequent treatment of this complex with triphosphate, ADP, ATP, or p₄A results respectively in the previously mentioned products. In similar reactions, Miller found that the stress protein LysU acts on ATP to form Ap₄A,⁸⁸ and Marx found that the ubiquitin activating enzyme UBA1 forms Ap₃A and Ap₄A.⁸⁹ Furthermore, introduction of a mixture of ATP and other NTPs resulted in unsymmetric Ap₄N analogues. Unsymmetric Ap₄N species have also been synthesized using luciferase as catalyst.⁹⁰ In an example that uses a genetically modified enzyme, Frey developed a modified human fragile histidine triad protein capable of catalyzing the reaction of NMP derived phosphorimidazolides and nucleotide substrates to make dinucleoside oligophosphates.⁹¹

Recent efforts have also shown potential utility for PPK enzymes that catalyze phosphoryl transfer from and to polyP. Jessen has shown that PPK2 not only catalyzes the transfer of one phosphoryl unit from polyP to ADP but can also competently utilize longer nucleotide substrates.⁹² In this manner, p₄A and p₅A were synthesized, with decreasing reactivity for longer nucleotides. Jendrossek then found that PPK2 from Agrobacterium tumefaciens is similarly capable of producing longer nucleotides with a greater distribution than the enzyme studied by Jessen, providing up to nucleoside nonaphosphates on an analytical scale.⁹³ PolyP binding to PPK2 has also been analyzed crystallographically, providing further insight into the mechanism of these reactions.⁹⁴

Biochemical Utilization of Oligophosphates

Oligophosphates are implicated in a variety of biochemical processes, resulting in use in assays and drug development. The most common oligophosphates are 5’-nucleotides, and p₄A and p₅A have been identified in a variety of biological systems. However, the full physiological impact of these extended nucleotides is not well known.^20–22 Nevertheless, several biological effects of p₄A have been identified such as reduction in rabbit intraocular pressure⁹⁵ and inducing vasodilation.⁹⁶ Adenosine 5’-pentaphosphate (p₅A) and uridine 5-pentaphosphate (p₅U) have also been shown to strongly inhibit Ribonuclease A, and these nucleotides may have similar effects on a myriad of enzymes.⁹⁷ Additional research may further elaborate the roles of extended nucleotides, but synthetic derivatives have found use in biochemical experiments as well. Most notably, nucleoside oligophosphates that have been terminally modified with fluorescent labels have been utilized in real time DNA sequencing.^98,99 The longer oligophosphates result in polymerase activity comparable to that of free NTPs, as opposed to the much slower reactivity of terminally modified NTPs.

Np_nNs have been relatively well studied, and Np_nNs as long as heptaphosphates have been identified in biological systems.^23,24 Physiologically, Np_nNs are potent agonists of many P2Y receptors, and these compounds have been investigated with respect to blood clotting.^43,102 For example, nucleotide pyrophosphatase/phosphodiesterase 4 (NPP4) was found to hydrolyze extracellular Ap₃A and Ap₄A, promoting platelet aggregation, and the authors suggested that development of inhibitors of this reaction may serve as antithrombotic drugs.¹⁰³ Uridine adenosine tetraphosphate (Up₄A) is also a vasoconstrictive agent, and it has been suggested as a potential treatment for cardiovascular disease.²³ A number of studies have also found Np_nNs to inhibit other enzymes such as adenylate kinase⁴⁵ and ribonucleotide reductase.¹⁰⁴ Denufosol, a Np₄N, has even been clinically tested as a treatment for cystic fibrosis, albeit with mixed results.²⁵ Another important area of study for Np₄Ns is in HIV drug resistance. The nucleotide analogs commonly used in the treatment of HIV act by terminating growing viral DNA when incorporated, due to absence of a 3’ hydroxyl group. Resistance to these drugs is conferred via a reaction in which the nucleotide analog is cleaved from the DNA terminus by ATP, generating adenosine nucleoside tetraphosphates (Ap₄N) and allowing reverse transcription to continue (Figure 6).^105,106 Research has shown that other Np₄Ns can be synthesized to inhibit this activity and potentially serve as drugs themselves via the reverse reaction.¹⁰⁷

Figure 6.

Simplified mechanism of HIV resistance to azidothymidine.

PolyP is ubiquitous in life and plays a variety of roles such as phosphate storage and part of blood clotting.^26,84 Biosynthesis of polyP has been studied in order to use microorganisms to recover phosphate from wastewater, a process termed Enhanced Biological Phosphorus Removal (EBPR).¹⁰⁸ However, research into the utilization of synthetically modified polyphosphates is scarcer. Several groups have sought to investigate proteins that bind polyP using biotinylated polyP. Jessen and Saiardi prepared a biotinylated octaphosphate to identify proteins in the human PolyPome,¹⁰⁹ and utilization of longer functionalized PolyP has been made possible through EDAC mediated coupling developed by Morrissey.¹¹⁰ Several groups have utilized biotinylated PolyP containing an average of 700 phosphate units per molecule to similarly screen or immobilize PolyP binding proteins,^110–112 and this material has been made commercially available through Kerafast. The same EDAC mediated reaction has also been used to modify PolyP with fluorophores to visualize PolyP transport into cells.³⁴ Another exciting development is the recent elucidation of protein polyphosphorylation as a nonenzymatic post translational modification, but it remains to be seen what new chemistry arises from investigation of this phenomenon.²⁸

Synthesis of Extended Oligophosphates

Monophosphorylation

We define monophosphorylation as a reaction in which an electrophilic monophosphate synthon reacts with a nucleophile. Therefore, treatment of such a monophosphorylation reagent with a phosphate substrate results in an oligophosphate product bearing one additional phosphate group. A number of syntheses of oligophosphates have been reported using this approach, as many of the monophosphorylation reagents are the same as those that have been utilized for the synthesis of shorter oligophosphates, such as NTPs.

A large class of monophosphorylation reagents is based on electrophilic activation of an NMP. In a representative example, treatment of an NMP with CDI results in a phosphorimidazolide species.⁴⁰ This imidazole group is a moderate strength leaving group that can be substituted by phosphate nucleophiles. A variety of additives have been used to accelerate these phosphorylation reactions and improve yields, commonly divalent metal salts such as MgCl₂ and ZnCl₂ as well as organic heterocycles such as 4,5-dicyanoimidazole (DCI).¹⁰⁰ Many reagents other than CDI have also been used to similarly activate NMPs, such as DCC,¹⁸ acylating agents,¹¹³ sulfonyl reagents,,¹¹⁴ and 2-imidazolyl-1,3-dimethylimidazolinium chloride.³³ Comparable phosphoramidate species have also been generated with various amine leaving groups such as morpholine¹¹⁵ and piperidine.¹⁰⁰ This strategy has been utilized extensively with NMP and NDP substrates to synthesize dinucleoside di-and triphosphates,^40,43,44,116 and extension of this methodology to longer oligophosphate substrates has given rise to a number of syntheses of extended oligophosphates.^{43,44,100,104} Rideout demonstrated synthesis of Up₄U through several routes including treatment of UMP derived phosphorimidazolide with pyrophosphate (Figure 7).⁴⁴ However, this synthesis provided only trace product contaminated by p₄U, demonstrating the inefficiencies of these reactions in the absence of a suitable promoter. In a more successful example, Wang synthesized p₄Ns in good yield from NMP derived phosphopiperidates utilizing DCI as a promoter (Figure 7).¹⁰⁰ Furthermore, the isolated p₄Ns were further monophosphorylated to produce Np₅Ns in moderate yield (Figure 7). Another approach to increase the reactivity of monophosphorylation reagents is to synthesize zwitterionic species with tertiary amine derived leaving groups, such as pyridine, but this strategy has so far only been applied to the synthesis of shorter oligophosphates.¹¹⁷

Figure 7.

Representative monophosphorylation syntheses of oligophosphates by Rideout,⁴⁴ Wang,¹⁰⁰ Kowalska,⁷⁰ and Shanmugasundaram.¹⁰¹

Monophosphorylation reagents are not limited to those derived from NMPs, and the above strategy can be applied to activation of other organomonophosphates.^70,98 However, electrophilic activation of inorganic orthophosphate presents a complication due to oligomerization.⁴¹ Accordingly, Kowalska reported a 2-cyanoethyl phosphorimidazolide, utilizing a protecting group widely used in oligonucleotide synthesis that is cleaved by mild base (Figure 7).⁷⁰ This reagent reacts readily with NDP and NTP substrates in a microwave assisted reaction to extend the phosphate chain by one unit. Notably this methodology also imparts a facile way to incorporate thiophosphates into the oligophosphate, a common strategy with shorter oligophosphates that has not been well developed for longer derivatives.

Phosphochloridates are also competent monophosphorylation intermediates. Treatment of nucleosides with phosphoryl chloride in alkyl phosphate solvents, the Yoshikawa method, results in 5’-phosphochloridate-nucleosides.⁵⁵ In the Ludwig synthesis of nucleoside triphosphates, these phosphochloridates are treated with pyrophosphate to generate a nucleoside-trimetaphosphate intermediate that is quenched with water to yield an NTP.¹¹⁸ In a comparable synthesis, Shanmugasundaram treated 5’-phosphochloridate-nucleosides with inorganic triphosphate, yielding p₄Ns in 40–48% yield (Figure 7).¹⁰¹

Jessen has also demonstrated a variety of P(III) based monophosphorylation reactions. A fluorenylmethyl protected phosphoramidite was used to selectively and iteratively extend nucleotides through a monophosphorylation reaction, providing syntheses of NDPs, NTPs, and p₄A.^71,72 The same methodology has been extended to a wider variety of substrates, providing access to thiophosphates, sugarnucleotide conjugates, and Ap₄U.¹¹⁹ Similarly, utilizing a phosphordiamidite reagent to couple two oligophosphate substrates generated products as long as Ap₇A.¹²⁰

Diphosphorylation

Diphosphorylation chemistry has not been as well explored as monophosphorylation, and these reactions are mostly limited to the synthesis of Np₄Ns. The reaction of an activating agent at only one terminal phosphate of pyrophosphate would be expected to result in oligomerization. However, treating pyrophosphate with an excess of activating agent may result in a pyrophosphate species bearing a leaving group at both ends. This chemistry is best represented by the work of Wright, in which a pyrophosphate salt is treated with carbonyldiimidazole (CDI) to form a bis-phosphorimidazolide (Figure 8).⁷⁹ This reagent reacts slowly with nucleoside monophosphates, a reaction that is accelerated with promoters such as ZnCl₂, to give symmetric Np₄Ns. This chemistry has not been demonstrated with longer substrates, such as nucleoside diphosphates, although this may be a route to dinucleoside hexaphosphates. Interestingly, the authors were able to synthesize similar diphosphorylation reagents from pyrophosphate analogs in which the bridging oxygen has been replaced with methylene derivatives, providing access to oligophosphates with nonhydrolyzable linkers.

Figure 8.

Representative Diphosphorylation syntheses of Np₄Ns.^44,62,79 Thermal ellipsoid plot of crystallographically characterized diphosphorylation reagent P₂O₅(DABCO)₂ with thermal ellipsoids set at 50% probability (red for oxygen, orange for phosphorus, blue for nitrogen, grey for carbon); this graphic was adapted from reference 62 as permitted under its CC BY 4.0 License https://creativecommons.org/licenses/by/4.0/.

A notable shortcoming of Wright’s diphosphorylation reagent is the difficulty of synthesizing unsymmetric Np₄Ns. Recently, syntheses of neutral, dizwitterionic adducts of P₂O₅ with N-donor bases such as DABCO and pyridine have been reported (Figure 8).⁶² These neutral N-donor bases are superior leaving groups as compared with the anionic phosphorimidazolides used by Wright. In a stark contrast in reactivity to Wright’s reagent, the pyridine adduct of P₂O₅ reacts instantly with AMP to give an adenosine substituted TriMP intermediate. Ring opening of this intermediate with UMP generates unsymmetric Up₄A in 75% yield. The extension of this methodology to longer oligophosphates similarly remains to be elucidated.

Diphosphorylation can also proceed through activation of a diphosphate compound. Similar to activation of NMPs, treatment of NDPs with CDI forms a terminal phosphorimidazolide, and this chemistry has been utilized to make symmetric Np₄Ns.^44,102 This methodology has so far suffered from low yields, and has only been utilized for the synthesis of symmetric Np₄Ns. In principle, it should be possible to treat an activated NDP with a different NDP or with inorganic pyrophosphate to synthesize unsymmetric Np₄Ns or p₄Ns, but this chemistry has not been developed. Meier has also activated diphosphate compounds with trifluoroacetic anhydride. However, this methodology has only been used in the synthesis of triphosphate derivatives.^122,123

Triphosphorylation

Activation of triphosphates results in an intramolecular dehydration to form cyclic TriMP derivatives. Therefore, triphosphorylation reactions proceed through a substituted TriMP intermediate, either through activation of a triphosphate substrate or directly from an activated TriMP reagent.

The traditional syntheses of NTPs by Khorana¹⁸ and Ludwig and Eckstein⁶⁰ proceed through nucleoside substituted TriMP intermediates. Aqueous hydrolysis of these compounds yields NTPs, but ring opening with other nucleophiles is possible to give a wide variety of terminally functionalized NTPs.^37,38 Ring opening can also be performed with phosphate nucleophiles to generate longer oligophosphates.^35,37 However, ring opening with a phosphate nucleophile generally requires a promoter such as a divalent metal ion. In one example, adenosine substituted TriMP formed through Ludwig-Eckstein methodology was ring opened with AMP and ADP to provide Ap₄A and Ap₅A (Jones, Figure 9).¹²¹ A prime drawback of starting with Ludwig-Eckstein chemistry is the need for protection of the 2’ and 3’ hydroxyl groups. However, it is also possible to generate nucleoside substituted TriMP intermediates directly from isolated NTPs by activation, for example with DCC. Compounds generated in this manner have then been ring opened with NMPs to make Np₄Ns^43,44 or with NDPs to make Np₅Ns.⁴⁵

Figure 9.

Representative triphosphorylation syntheses of oligophosphates by Jones,¹²¹ Taylor,³⁵ and Jessen.³⁷

In recent years, several TriMP based reagents have been developed to directly triphosphorylate a nucleophile rather than starting with an NTP substrate. Initial investigations attempted direct reactions of TriMP with NMPs, with some success generating p₄Ns.¹²⁴ However, this methodology produces a distribution of nucleoside oligophosphates, hampering its utility. The first electrophilic activation of TriMP comes from Taylor, who in situ activated TriMP with mesitylenesulfonyl chloride and nucleophilic tertiary amines,^35,59,65,66 a reaction which has also been utilized by Kool.¹²⁵ TriMP has been activated similarly with (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, (PyAOP), and in this case the resulting activated TriMP derivative could be structurally characterized.⁶¹ Furthermore, it was reported subsequently that a derivative of TriMP activated with a sulfonyl chloride in direct analogy to Taylor’s procedure could be isolated and structurally characterized.⁶² Jessen^37,38 and Huang⁶⁷ have taken a related approach of synthesizing mixed P(III)-P(V) TriMP analogs that similarly react with nucleophiles to give substituted TriMP species. In one example, Taylor synthesized p₄Ns and Np₄Ns by treatment of activated TriMP with an NMP and ring opening of the resulting substituted metaphosphate either with water or another NMP (Figure 9) As a P(III) species, Jessen’s triphosphorylation reagent is highly reactive towards even difficult substrates such as NTPs. In addition to a number of other triphosphorylation reactions, Jessen has demonstrated triphosphorylation of ATP and ring opening of the subsequent TriMP intermediate to make a rare p₆N derivative (Figure 9).³⁷

Tetraphosphorylation

One of the few examples of a tetraphosphorylation reagent is [PPN]₂[P₄O₁₁], the anhydride of dihydrogen tetrametaphosphate (TetraMP, Figure 10).¹²⁶ This isolable and crystallographically characterized reagent has been treated with nucleosides and NMPs to make substituted TetraMP intermediates. Subsequent ring opening with hydroxide yielded p₄Ns and p₅Ns, which were studied as inhibitors of RNase A (Figure 10, top).⁹⁷ In a follow up report, [PPN]₂[P₄O₁₁] was further shown to be a competent tetraphosphorylation reagent for a variety of other nucleophiles and ring opening reactions, yielding a diverse class of tetra- and pentaphosphate derivatives, including an unusual example of a tetraphosphorylated amino acid, tyrosine.⁴⁶

Figure 10.

A. Tetraphosphorylation synthesis of p₄Ns.⁹⁷ B. Synthesis of a phosphonate analog of a 3’,4’ didehydronucleoside.⁴⁶ C. Thermal ellipsoid plot of tetraphosphorylation reagent ([PPN]₂[P₄O₁₁]) with thermal ellipsoids set at 50% probability and cations omitted for clarity (red for oxygen, orange for phosphorus).¹²⁶ D. Tetraphosphorylation reaction utilizing P(III) oligophosphites by Parang.¹²⁷

In an unusual example of C-phosphorylation, a previously reported TriMP based triphosphorylation reagent was used to demonstrate triphosphorylation of the phosphorus ylide methylenetriphenylphosphorane.⁶¹ Similarly, treatment of [PPN]₂[P₄O₁₁] with methylenetriphenylphosphorane results in C–P bond formation followed by deprotonation to form a new phosphorus ylide (Figure 10, bottom).⁴⁶ This ylide further reacts with aldehydes through Wittig chemistry and is therefore capable of generating tetraphosphate analogs where the terminal P–O bond has been replaced with a non-hydrolyzable olefinic P–C bond. In a particularly interesting example, treatment of the tetraphosphorylated ylide with a protected uridine derived aldehyde results in a tetraphosphate/phosphonate of a 3’,4’-didehydronucleoside (Figure 10, bottom).

Some tetraphosphorylation chemistry has also been developed with P(III) chemistry. In an unusual strategy, Parang developed a methodology to prepare oligophosphites, P(III) analogs of oligophosphates. Via iterative coupling and hydrolysis reactions of P(III) reagents, di-, tri-, and tetraphosphites were synthesized. These oligophosphites were then bound on a polymer support and treated with nucleosides. Subsequent oxidation and deprotection yielded symmetric Np₂Ns, Np₃Ns, and Np₄Ns.¹²⁷

Pentaphosphorylation and Beyond

A variety of monophosphorylation strategies exists, but the prevalence of methodologies decreases steeply for diphosphorylation and beyond. This is potentially explained by the comparatively rarer known applications for extended oligophosphates, the difficulty of obtaining larger phosphate starting materials, and the more difficult purification of extended oligophosphates. Nevertheless, there is a nascent field of phosphorylation reagents based on tri-^{35,37,38,59,61,65,66} and tetrametaphosphate.^46,97,126 Larger metaphosphates, up to at least dodecametaphosphate, are fairly easily synthesized via flux methods,¹² and we can naturally expect related chemistry derived from larger metaphosphates in the future. This may take the form of activation of these metaphosphates, similar to the work of Taylor^35,59,65,66 and Cummins,^46,61,97,126 or mixed P(III)-P(V) reagents like those of Jessen^37,38,128 and Huang (Figure 11).⁶⁷ Achieving high selectivities and yields with these larger reagents may prove difficult, but these routes seem promising for rational synthesis of well defined oligophosphate chain lengths.

Figure 11.

Proposed penta- and hexaphosphorylation reagents in analogy to the crystalline tetraphosphorylation reagent [PPN]₂[P₄O₁₁]^46,97,126 and proposed mechanism of EDAC mediated functionalization of PolyP.

The scant methods for polyphosphorylation are not typically selective for a specific chain length. Rather, distributions of polyP starting materials are used, giving rise to a similar distribution of difficult to separate products. A variety of formulations of polyP are commercially available, often denoted as “hexametaphosphate” or Graham’s salt, usually produced by thermal condensation of lower phosphates.^129,130 The distribution of phosphate chain lengths varies considerably and can be roughly analyzed by phosphorus NMR integration or SDS-PAGE.¹³¹ More precise quantifications of these distributions, perhaps via MALDI-TOF or capillary electrophoresis, remain desirable. Depending on the synthesis and supplier, these polyP formulations range in average phosphate chain length from P12 to >P500 and often contain large quantities of metaphosphates and short oligophosphates.

The polyP chemistry developed thus far has been aqueous. Aqueous systems prevent the use of water reactive species commonly used in phosphorylation chemistry, but aqueous media also provides the opportunity to phosphorylate biomolecules such as peptides and unprotected amino acids that are only appreciably soluble in water. Most of the known polyphosphorylation chemistry is based on Morrissey’s activation of polyphosphate with aqueous EDAC.¹¹⁰ The initial report proposes activation of the terminal phosphate units of PolyP to generate isourea leaving groups, which are intercepted by amine substrates to form terminally phosphoramidate modified polyphosphate.^110,132 However, O-phosphoryl-isourea species are known to rapidly isomerize to N-phosphoryl-urea species which do not act as strong leaving groups.⁴¹ Therefore, we propose a modified mechanism involving EDAC mediated intramolecular dehydration (Figure 11). The resulting ultraphosphate species would then go on to react with nucleophilic substrates resulting in the final functionalized polyP. Also noteworthy is the fact that this functionalization is expected to occur at both ends of the polyP. This EDAC promoted polyphosphorylation procedure has since been expanded towards alcoholic substrates¹³³ and been used in sporadic biochemical studies.^34,111,112 Although activation of p₃Ns has been widely used in triphosphorylation protocols,^43–45 a similar activation strategy has not been applied to well defined extended oligophosphates. For example, treatment of p₄Ns or p₅Ns with EDAC may provide an effective means of tetra- and pentaphosphorylation.

Polyphosphorylation of peptides has also recently grown in interest as an analog of protein monophosphorylation, the most common post translational modification.¹³⁴ The last several decades have seen debate and investigation of how protein pyrophosphorylation fits into the canonical understanding of protein phosphorylation,¹³⁵ and in 2015 Saiardi added the discovery of protein polyphosphorylation to this discussion.²⁸ Polyphosphorylation was found to occur on lysine residues via a nonenzymatic reaction, and the authors went on to develop a yeast model to further study this post translational modification.¹³⁶ The mechanism, importance, and pervasiveness of protein polyphosphorylation all remain areas of necessary study.

Outlook

The chemical synthesis of oligophosphates is simultaneously complex and nascent. Despite being a relatively small class of molecules, hundred of papers and multiple reviews detail the syntheses of nucleoside mono-, di-, and triphosphates.^17,19,54 Whether due to difficulty of their synthesis or less demand for their applications, the synthesis of extended oligophosphates has certainly lagged behind that of their shorter analogs.

Further advances in the synthesis of p_nNs and Np_nNs are still possible to ease synthesis and purification, but a fair number of preparations of these compounds has been reported. Future synthetic work should endeavor to push past these compounds to more complex and varied products. A prime future direction should be the investigation of 3’-nucleotides. The biorelevance of such compounds is less well developed, but 3’-nucleoside oligophosphates have potential to interact with enzymes that bind DNA/RNA due to the 3’ linkages in these biopolymers. The synthesis of 3’-nucleotides also represents a somewhat greater challenge than analogous 5’-nucleotides due to the lower steric accessibility of this site, and the potential interference or hydrolysis promoted by the 2’ hydroxyl group. Another area for further exploration is nucleotide analogs derived from non ribose sugars, such as TNA triphosphates, which have been synthesized by Chaput.^73,137 Dinucleoside oligophosphates similarly suffer from a dearth of 3’,3’ or mixed 3’,5’ species. One could even envision a cyclic product in which the 3’ and 5’ positions of a single nucleoside are bridged by an oligophosphate. Such cyclic pyrophosphate derivatives are reminiscent of pyrophosphate linked DNA¹³⁸ and cyclic dinucleotides.¹³⁹

Greater modification of the oligophosphate itself is also desirable. A significant class of ATP analogs are those in which the final bridging oxygen of the oligophosphate has been replaced with a non-hydrolyzable group, such as a methylene.¹⁴⁰ This blocks hydrolysis to ADP, inhibiting enzymes and allowing biochemical study of their mechanisms. Such modifications are fairly common with shorter oligophosphates³⁸ but are limited to a few examples for longer phosphates.⁷⁹ Additionally, formation of thiophosphate analogs have not been reported for many large oligophosphates,⁷⁰ despite thiophosphates being the basis for many drugs.¹⁴¹

A significant recent development in oligophosphorylation is the introduction of well-defined isolable oligophosphorylation reagents. The earliest oligophosphorylation procedures, such as Khorana’s synthesis of ATP,¹⁸ rely on in situ activation of nucleotides and inorganic phosphates. In the case of Khorana’s synthesis, the favorable formation of TriMPs results in a viable NTP synthesis, but the prevalence of side products results in low yield and difficult purification. In contrast, researchers have now developed bottleable reagents to append oligophosphates to a desired substrate.^{37,38,46,61,97,126,128} By introducing an activated phosphate reagent to a substrate of choice, this strategy reduces the potential side reactivity caused by activation of a phosphate substrate. For example, CDI activation of NMPs is an effective procedure,⁴⁰ but there is still potential to form a 3’,5’-cyclic NMP side product. Utilizing a pre-synthesized (and potentially commercially available) reagent therefore simplifies the procedure and mitigates side reactivity.

Lastly, this Perspective has focused heavily on nucleosideoligophosphate conjugates and biochemical applications of these compounds, as this area has been the most developed. However, nucleotides are only a subclass of oligophosphorylated organic molecules, and there is potential for these compounds in a variety of fields of study and industry. For example geranyl diphosphate is a critical intermediate in terpene synthesis,¹⁴² biotinylated oligophosphates have been used in a number of studies,^109–112 and neither system contains nucleoside moieties. A much less developed but promising area is the utilization of oligophosphates in materials. A number of porous materials have been developed from zirconium phosphates and phosphonates,^143–145 but the utilization of oligophosphates for such applications has not to our knowledge been explored. Similarly, many phosphate and phosphonate containing polymers have been developed for biomedical or ion exchange purposes,¹⁴⁶ but oligophosphates remain essentially unexplored. However, the field of chemical oligophosphorylation has flourished, providing a variety of simple syntheses of not only di- and triphosphates but extended oligophosphates as well. By introducing this chemistry to a wider audience, we hope this Perspective will stimulate further interest and study of extended oligophosphates, in the biochemical field and potentially far beyond.