Notes & Tips Quantitative single-step purification of dinucleoside polyphosphates Michael Wright, Julian A. Tanner, and Andrew D. Miller * Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, South Kensington, London SW7 2AZ, UK Received 19 September 2002 5 0 ,5 000 -P 1 ; P 4 -Dinucleoside polyphosphates are an im- portantfamilyofnucleotideswithbothintracellularand extracellular biological roles [1,2]. Accordingly, there have been a number of synthetic methods reported for the preparation of dinucleoside polyphosphates, allow- ing these biological roles to be studied in more detail. One of the most successful methods involves the use of the heat-inducible Escherichia coli lysyl-tRNA synthe- tase (LysU) 1 as a flexible enzyme catalyst for the syn- thesis of a wide range of dinucleoside polyphosphates using purified adenosine 5 0 -triphosphate (ATP) with a second nucleotide as substrate [3]. LysU biosynthesis of dinucleosidepolyphosphatesisanefficientalternativeto multistep chemical syntheses [4]. Traditionally, thin- layer chromatography (TLC) has been used to monitor reactionsandsubsequentpurificationscarriedoutusing reversed-phase high-performance liquid chromatogra- phy(HPLC).WehaveconsistentlyobservedthatTLCis frequently unreliable and gives poor quantitative anal- yses. Furthermore, reversed-phase HPLC poorly re- solves dinucleoside polyphosphates with similar properties. Accordingly, we have encountered frequent problems in the isolation and purification of dinucleo- side polyphosphates and their analogues, limiting our attemptsatcharacterizingtheirbiologicalactivity.Here we report a fast (<5min), quantitative method of di- nucleoside polyphosphate analysis and purification that is reproducible and has solved all our basic problems with TLC and HPLC analysis/purifications. This method is a single step ion-exchange technique that we have found to give high-resolution purification withouttheneedforsubsequentdesaltingofthepurified product. By way of illustration, we describe the updated syn- theses and subsequent purification of three different di- nucleoside polyphosphates: b; c-methylenediadenosine 5 0 ,5 000 -P 1 ; P 4 -tetraphosphate (Ap 2 CH 2 p 2 A), diinosine 5 0 ,5 000 -P 1 ; P 4 -tetraphosphate (Ip 4 I), and b; c-methyl- enediinosine 5 0 ,5 000 -P 1 ; P 4 -tetraphosphate (Ip 2 CH 2 p 2 IÞ: Ap 2 CH 2 p 2 A was prepared by means of the LysU bio- synthesisprocedureupdatedfrompreviously[3].Briefly, LysU (30 lM, dimer) was dialyzed at 4 °C against 50mMTris–HCl,pH8.0(1L)andlefttoreactivatefor a minimum of 48h. Reaction mixture (4ml) was then prepared consisting of 9 lM LysU, 2mM L -lysine, 10mM MgCl 2 , and 75 lg yeast inorganic pyrophos- phatase(Roche)in50mMTris–HClbuffer,pH8.0.This mixture was incubated at 30 °Cwithstirringand5mM ATP,7.5mM b; c-methylene-ATP (AMP-PCP, Sigma), and 160 lM ZnCl 2 was slowly added. Incubation was continued for a further hour or until the formation of Ap 2 CH 2 p 2 Awasjudgedtobecomplete.Ip 2 CH 2 p 2 Iwas prepared from Ap 2 CH 2 p 2 A and Ip 4 I directly from Ap 4 A (Sigma) using the enzyme 5 0 -adenylic acid deam- inase from Aspergillus (Sigma). This unspecific en- zyme is able to deaminate adenosine nucleotides to their inosine counterparts [5]. Our updated procedure was to prepare a reaction mixture (2ml) containing 5 0 - adenylic acid deaminase (0.22U) and diadenosine polyphosphate (20mM) in 50mM Tris–HCl, pH 6.5. Thismixturewasincubatedat35 °Cfor1hwithstirring or until the formation of product was judged to be complete. In all cases reaction progress could be closely moni- toredusingaResourceQanion-exchangecolumn(6ml), connected to a fast protein liquid chromatography (FPLC) system equipped with UV lamp (254nm) and Analytical Biochemistry 316 (2003) 135–138 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: +44-20-7594-5803. E-mail address: a.miller@imperial.ac.uk (A.D. Miller). 1 Abbreviations used: LysU, lysyl-tRNA synthetase; Ap 2 CH 2 p 2 ,A, b-c-methylenediadenosine 5 0 ,5 00 -P 1 ; P 4 -tetraphosphate; Ip 4 I, diinosine 5 0 ,5 000 -P 1 ; P 4 -tetraphosphate; Ip 2 CH 2 p 2 I, b-c-methylenediinosine 5 0 ,5 000 - P 1 ; P 4 -tetraphosphate; FPLC, fast protein liquid chromatography; TEAB,triethylammoniumhydrogencarbonatebuffer;FAB,fastatom bombardment; ES, electrospray. 0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00035-6