Analysis of Organophosphorus Compounds. 1. Application of Iodine-Azide Reaction for Detection of Thiophosphoorganic Compounds in Thin-Layer Chromatography

A C T A U N I V E R S I T A T I S L O D Z I E N S I S

FOLIA CHIMICA 1 3 ,2 0 0 4

ANALYSIS OF ORGANOPIIOSPHORUS COMPOUNDS. 1. APPLICATION OF IODINE-AZIDE REACTION FOR DETECTION

OF THIOPHOSPHOROORGANIC COMPOUNDS IN TIIIN-LAYER CHROMATOGRAPHY

by Andrzej K otyński“, Zbigniew H. K udzin*1’ and W itold C iesielski1’

"D ep a rtm en t o f B io in o rg a n ic C hem istry, M ed ica l U niversity o f Łódź, / M u szyń skieg o Sir., Ł ó d ź 90-151, P oland

'’In stitu te o f C hem istry, U niversity o f Łódź, 6 8 N a ru to w icza Str., L udź 90-136, P oland

The application o f organophosphorus com pounds as inducing agenis in (lie iodine- azide reaction was investigated. Their induction activity w as exhibited by thiophosphoryl compounds; their induction coefficien ts were dependent on the number and nature o f sulphur atoms in the P(S)„ function. T hese relationships can be used for the group differentiation o f organophosphorus com pounds, for exam ple phosphates, thiophosphates and dithiophosphates. On the basis o f their induction activity, thiophosphoryl inductors determination m ethods (titrimetric, coulom etric and spectrophotometric methods) based on determination o f the quantity o f consum ed iodine (/xmol to nmol scale), was elaborated. The correlation betw een induction factors (Fj) in the iodine-azide reaction and detection lim its (D L S) using the iodine-azide reagent has been established. The iodine-azide reagent has been used for the selective thin-layer chromatographic detection o f several phosphorothioates, including sugar and/ nucleosides phosphorothioates and related com pounds. Comparison o fT L C detection system s for phosphorothioates using iodine-azide procedures and other representative procedures are presented.

K ey w o rd s: induced iodine-azide reaction; organophosphorus inductors; phosphorothioates; sugar phosphorothioates; thiophosphoryl nucleotides; micro­ determination o f thiophophoryl inductors; detection; TLC.

1. Introduction

Thiophosphoroorganic com pounds represent an abundant and structurally diverse group o f organophosphoro-derivatives o f great industrial

[1-4 | and synthetic im portance [5,6,7]. Structural form ulae o f the basic types o f thiophosphoroorganic com pounds are presented in Table 1.

Table 1. B asic types o f thiophosphoroorganic compounds.

No Structure T ype o f

com pounds“ N o Structure

T ype o f com pounds“ 1 R3—0 R 2 - 0 - ' p = 0 R '—o ' Phosphates 11 R3—S R 2 - S - V = S R’ —S Phosphoro-tetrathiolates 2 R3—O R 2 - 0 - ' p = 0 R’ —S Phosphoro-thiolates 12 R3—0 R 2 - 0 - P = 0 r/ Phosphonates 3 R3—0 R 2 - 0 - ' p = S R’ - O Phosphoro-thionates 13 R3—0 R 2 - 0 - P = S r/ Phosphono-thionates 4 R2— 0 R t - 0 - P = S / R2N Thiophosphor -amidates 14 r2n^ R N — P =S r/ Diam idothio-phosphonates 5 r2n r2n—p=s R’— ® Thiophosphor -diamidates 15 R3—0 R2—V = S R' Phosphino-thionates 6 _{R2—o j ? ^ 0 — R2} R' — R1 M onothiopyro -phosphates 16 R2N\ R2— P = S r/ Am idothio-phosphinates 7 R2— O^fi ^ 0 — R2 R'— O ^ O ^ O —R’ Dithiopyro-phosphates 17 R3— S R2— P = S R’ Phosphino-dithionates 8 R3—O R2- 0 - V = S R ' - S Phosphoro-thiolothionats 18 _{R2s J f i / R 2} R’ ^ p ^ s - s r " p^ R ' Dithiophos-phinates disulfides 9 _{R2- 0 , J Lo- R 2} r' -o''p''s- s^p''o-ri Dithiophos-phates disulfides 19 R\ R2 — P = 0 R1 Phosphine oxides 10 R3—0 R2- S —V = S R ' - S Phosphoro-trithiolates 20 R\ R2 — p = S / R1 Phosphine sulfides

"Acids: n (R 1 = R2 = R1 = H ); nA (R 1 = R2 = H); nB (R 1 = H); nC (R ï H); a (M e): b (Et); c (Bu); d (H ex); e (Oct); f (Ph); etc.; [e.g. 1 = phosphoric acid; 1A = alkyl (or aryl, or aralkyl) phosphate; IB = dialkyl phosphate; 1C = trialkyl phosphate; and correspondingly - IC a = trimethyl phosphate].

Follow ing S chroder’s discovery o f insecticidal properties of thiophosphoroorganic com pounds in the 1930’s [8,9], the num ber of thiophosphoroorganic insectides has been grow ing continuously L8-12]. ll is estim ated that, to date, over 500 different thiophosphoroorganic derivatives have been synthesised and tested over 100 o f which are com m ercially available as plant protection agents. Such dynam ic developm ent o f this agrochem istry dom ain presents an indicator o f the effectiveness o f thiophosphoroorganic insecticidals. M oreover, unlike chloroorganic com pounds, thiophosphoroorganic do not accum ulate in the environm ent and their fast biodégradation m akes them environm entally-friendly [12].

Structurally varied thiophosphoroorganic com pounds exhibit diverse biological activity and apart from being used as insectidals they have found other agrochem ical applications 112]. Thus, thiophosphoroorganic com pounds are widely used as insecticides (Table 2), acaricides, nem atocides, fungicides, bactericides, herbicides, rodenticides, growth regulators, insect chem osteriiants and insecticide synergists. Several thiolophosphates [(R 0 )2P (0)S ~ | were found as very useful tool in exploring enzym atic reaction m echanism s since they react considerably slow er than their oxygen analogs.

Table 2. Representative thiophosphoryl insecticides [1 1,12] Thiophosphoryl insecticides

Structure Name Structure Name

M e " 0 , ,,S M e^ 0 ' P ' ~ S - C - C ( 0 ) 0 E t C —C (0 )0 E t H2 Malathion M e " ° , 0 M e ^ s ^P ^ S - C - C ( 0 ) 0 E t C - C ( 0 ) 0 E t H2 lso-Malathion M e " ° / S M e ^ 0 ' P " S - C - - C ( 0 ) N H M e Dimethoate M e " 0 , , 0 p M e ^ ' " N H 2 O Methaamido-phos M e '0 , ,S _Fenthion Me"0, ,S p Me^ ' \ —s 0 Me no2 Fenitrothion iPr- ° y O iPr 0 , P ' s — Bz Iprobenfos M e " ° s , 0 M e" S ' P " H _A C Acephate Et— 0 s > : P h - S Et Fonofos E t - 0 o A Et—0 S - C - S - E t h2 Phorate

B iological activity of phosphorothioates is attributed to their thiono- thiolo isom érisation (Fig. 1) occurring in aqueous solutions.

R" ° y P _ _ R " ° v ,,S

rV P's- h r' o' P'0 - h

Fig. 1. T hiono-thiolo rearrangement

M ore recently, phosphorothioate analogues o f num erous biophosphates, such as phospholipids (Table 3), sugars phosphates (Fig. 2) or nucleotides (Fig. 3), were synthesized and used as im portant tools for basic research in biochem istry and m olecular biology.

Thiophospholipids (Table 3), isosteric with natural phospholipids were found to exhibit diverse pharm acological activity, including in anticancer, antivirus, antipyretic, antiallergic and im munom odulatory area [ 13,14 |. These com pounds also play an im portant role in the investigations on a polym orphism o f phospholipid based bio-m em branes [13,15,16] and also in enzym e action m echanism s o f the phospholipase class [17-19].

Table 3. Main types o f thiophospholipids (exem plified by l,2-d istearylo-3-thiophos- phatydylolipids) (X: O or S; Y: O or S) T h io p h o sp h o lip id s S tru ctu re R N am e Me3N -C H 2CH— l,2-distearylo-3-thiophosphatydylocholine o ° ' o HO—CH2CH— l,2-distearylo-3-thiophosphatydylo-ethanoloamine HO—^— (j!— NH2 l,2-distearylo-3-thiophosphatydylo-serine x .F 1 0 -0 -0 ■“ I I I o o 1 X X l,2-distearylo-3-thiophospliatydylo-glyeerol H2< f -HC-OH c — h2 l,2-distearylo-di-3-thiophosphatydylo-glyeerol l,2-distearylo-3-thiophosphatydylo-m yo-inositol

Sugar phosphorothioates and/or phosphorodithioates are applied as anti­ glaucom a and/or anti-parasite drugs [20,21] and also as plant protection agents [22]. Sugar phosphorothioates are also used as tools in investigations o f enzym atic reactions m echanism s [23,24],

~ 0 H R ' - 0 P -~ O H H OH _ O H B '-0 ~ O H

X: O or S; Y: O or S; R, R': H, alkyl, aryl or aralkyl m oiety

Fig. 2. Structures o f representative phosphorothioates o f sugars exem p lified by g lu cose (A ) and d eoxyglu cose (B ) derivatives.

B: purine or pirim idine base; X; H or OH; Y: O or S; Z: O or S; R, R ’, R ” : alkyl, aryl or carbonate moiety; n: polym erisation degree

Fig. 3. Structures o f phosphorothioate analogues o f nucleotides and oligon ucleotides (polinucleotides).

Phosphorothioate analogues of nucleotides and oligonucleotides, introduced originally by Eckstein [25], have found wide application in both biochem istry [26] and m olecular biology [21]. Recently, a considerable interest is observed in oligonucleotides as potential antisense m odulators o f gene expression [28-31], Thus, phosphorothioate analogues o f oligonucleotides were

found to be good candidates for antiviral and/or antitum or drugs (antisense strategy) [28].

Som e thiophosphoroorganic com pounds such as VX |O -ethyl-S-(2- diisopropylam ino-ethyl)-m ethylthiophosphonate], (he most lethal nerve gas agent ever created, may be used as chem ical weapons [32].

R = Me, Et; R" = iPr VX

Fig. 4. Structure o f V X type agents

Since the entry into force in 1997 o f the Chem ical W eapons C onvention (CW C), which requires ratifying countries to destroy stockpiled chemical w eapons, a need has arisen to implement analytical procedures to m onitor the process [32],

A wide array o f dialkylphosphorodithioic acids salts - particularly zinc salts [(RO)2P(S)S ]2Zn - are used as anti-w ear and anti-corrosion lubricating oil additives. These salts react with metal surfaces o f gears and m oving engine parts to im prove sm oothness and provide excellent resistance to rust and corrosion. Various applications o f dialkylphosphorodithioic acids salts [R O )2P (S )S ] were reported in detail by Plaza [33], The sodium salt, (iPrO )2P(S)SN a, is used as an activator in low -tem perature vulcanisation of rubber products [34]. Am m onium and sodium salts o f dialkylphosphorodithioates (R = Et or iPr) are used as flotation agents to suspend and separate m etallic ores from unwanted contam ination [34], A ntim ony tris 0 ,0 -d ialk y lp h o sp h o ro d ith io ates, l(R O )2P(S)S J^Sb, are used as passivating agents in petroleum refining since they prevent the poisoning ot the catalyst by contam inant m etals which are present in oil feeds [34,35],

2. Analysis o f Organothiophosphorus Compounds

Due to num erous applications of thiophosphates on the one hand and difficulties with their analysis [36] on the other hand, it is crucial to im prove their determ ination m ethods in terms o f selectivity and sensitivity. In spite o f many publications on the subject, quantitative and qualitative determ inations of the m ajority o f organothiophosphorus derivatives still pose a challenge for contem porary analysis. Up to now these com pounds have been analysed chiefly using chrom atographic m ethods with only a narrow range o f other techniques [37,38].

Phosphorothioates have been determ ined by quantitative TLC followed by brom om etric titration [39], voltam m etric determ ination [40] or densitom etric determ ination preceded by reaction with palladium reagent [41]. Phosphorothioates as well as phosphorodithioates have been determ ined by means o f acidim etric titration after reaction with chlorom ethylpyridinum iodide [42], potentiom etric titration using ion-selective electrodes [43], argentom etric [4 4 ,4 5 1 or m ercurom etric titration [46] and by polarographic m ethods [47], Phosphorodithioates have also been determ ined by atom ic absorption spectroscopy o f their cupric(ll) com plexes [48,49], Several types o f thiophosphoryl com pounds, including phosphine sulfides, phosphorothioates and phosphorodithioates have been determ ined spectrophotom etrically on the basis o f their charge-transfer com plexes with tetracyanoethylene (TCNE) or tetracyanoquinodim ethane (TCNQ) [50-52], Phosphorothioates have been determ ined by chem ilum inescence method [53], and by ion-exchange chrom atography coupled with electroconductive or spectrophotom etric detectors |54J. The analytical determ ination o f some thiophosphorus insecticides (m etham idophos, iso-m alathion, fenitrothion) using a coulom etric titration with the anodically generated chlorine and biam perom etric end-point detection was elaborated by Ciesielski and co-w orkers [55], Phosphorothioates and phosphorodithioates have also been selectively detected using im m unoassay [56], T hiophosphoryl com pounds are also analized using Gas C hrom atography [57], Mass Spectrom etry [58], Flow -lnjection-A nalysis, A tom ic A bsorption Spectrom etry [59], and also by Fourier-transform Raman Spectroscopy [60].

2.1. Analysis of Organothiophosphorus Compounds Using UP NMR

A lthough " P NM R spectra were reported as early as 1951 it was the availability o f com m ercial m ultinuclear NMR spectrom eters by 1955. That led to the application o f 3IP NMR as an im portant tool for structure elucidation. W ith the introduction by 1970 o f Fourier-transform (FT) and high-field superconducting m agnet NMR spectrom eters, ' 1P NMR spectroscopy expanded to the study o f biological phosphorous com pounds. The UP nucleus has convenient properties suitable for FT NMR: spin Vi, 100% of natural abundance, m oderate relaxation time and a wide range o f chem ical shift [5( UP) > 600 ppm], which recom m end this technique for organophosphorus com pounds analysis. W ith substantial increase o f sensitivity o f m odern FT N M R spectrom eters, analytical applications o f this technique grew dram atically during the last decades [61].

For analysis (identification and/or determ ination) o f phosphorus- containing com pounds, including thiophosphoryl derivatives, !IP NMR technique is w idely used in analytical as well as organic and related chem istry

f61,62j. T he analytical potential of this technique is reflected by results com piled in T able 4.

Table 4. Representative ' T NMR spectra o f phosphorus com pounds [62]

No Compound structure 8 (J1P) [p p m ]a No Compound structure ft(" P ) I ppm] “ 1 h,p o4 0.0 21 (EtO)3P=S 68 2 N aH 2P 0 4 0.5 22 (EtS)(EtO )2P=S 94 3 N a ,H P 0 4 3.5 23 (EtO )jP(S)-SH 86 4 N a ,P 0 4 6.0 24 [(EtO)2P(S)-S-]2 84 5 N a3P 0 3S 32 25 (E tS)3P=S 92 6 N a3P 0 2S2 61 26 HjP Oj 5 7 N a3POS3 86 27 NaH 2P 0 3 4 8 N a3PS4 87 28 N a2H P 0 3 4 9 ( E t 0 ) 3P = 0 0.0 29 (E tS)3P 115 10 (E tS )(0 E t)2P = 0 26 30 (EtO)jP(S)H 61 11 (E tS)2(0 E t)P = 0 54 31 (E t 0 ) 2P (0 )H 7.5 12 (E tS)3P = 0 61 32 (EtO)2P(S)SH 67 13 M e3P = 0 36 33 Me.,P=S 59 14 Bu3P = 0 41 34 Bu3P=S 48 15 Ph3P = 0 25 35 Ph3P =S 40 16 M e -P (0 )(0 E t)2 30 36 M e-P (0 )(S E t)2 57 17 M e-P(S)(O E t)2 95 37 M e-P(S)(SE t)2 78 18 M e2(PrS)P=S 53 38 (H 2N ).,P =0 22 19 Et2(PrS)P=S 61 39 (M e2N )3P = 0 23.5 20 (M e2N )3P=S 81 40 (H 2N )3P=S 61

'' Positive chem ical shift values are reported for com pounds absorbing at lower fields than H3P 0 4.

T he dependence o f chem ical shifts o f " P nuclei in acidic organophosphorus com pounds on pH (Table 4, [63-65]), causes that supplem entary techniques in their analysis (including T LC supplied with selective detection) are required.

2.2. Thin-Layer Chromatographic Detection of Organophosphorus Compounds

T hin-layer chrom atography (TLC) com bined with chem oselective detection has been considered as the m ethod o f choice, especially for non­ volatile and therm ally unstable organophosphorus derivatives [37-40,66,67j. Thus, phosphorothioates and phosphorodithioates have been detected by TLC using silver nitrate solution alone [68] or in conjunction with chelating indicators (e.g., brom ocresol green) [68-70] and using copper(II) chloride solution and potassium hexacynoferrate(III) solution [71 ] as subsequent spray reagents. Also, potassium iodoplatinate [38], palladium (II) chloride [38,41,72] and palladium (II) com plexes with fluorescent indicators [e.g., palladium !II)-calcein] [73] have been w idely used for the detection o f thiophosphoryl insecticides. Phosphinothioate metal com plexes have been localized on TLC plates by means o f a dithizone reagent [40,74], The detection o f thiophosphoryl com pounds has also been achieved using 2,6-dibrom o-benzoquinone-4-chlorim ine (DCQ) [75,76], an am m onia solution o f 4-m ethyl um belliferone preceded by brom ine vapour treatm ent [76], fluorescein [77], am m onium m olybdate reagent [71,78,79] and potassium iodate solution [80] as spray reagents.

The thiophosphoryl com pounds are visible in the UV region (254 nm) using fluorescent-chrom atoplates [81 ]. Several reports have described the use of the T L C -enzym e inhibition (TLC-E1) technique for the detection o f a variety o f organophosphorus com pounds, including the P-S type com pounds [38,82-84].

M ost o f the procedures cited above seem to em ploy general rather than specific detection reagents for thiophosphoryl com pounds therefore we turned our attention to the iodine-azide reaction which is known to be induced by various sulfur com pounds.

3. Application of the Iodine-Azide Reagent

lodine-azide reagent has been used in the analysis o f divalent sulfur com pounds for a num ber o f years after the iodine-azide reaction was first described by R aschig at the beginning o f the twentieth century [85]. In the course o f the induced iodine-azide reaction iodine is consum ed and nitrogen is evolved [86],

S(II)

12 + 2N 3~ — ---21 + 3N2

S(II) - inductor containing sulfur (II) Pig. 4. General schem e o f the induced iodine-azide reaction

T he sensitivity o f sulfur com pounds determ inations heavily depends on the induction coefficient defined by the equation (1):

where: n, is m oles o f iodine consum ed in the induced reaction and n, is moles of the inductor.

This im plies straight forward relationship - the higher the induction coefficient, the m ore sensitive the determ ination o f a given inductor [86-95].

3.1. Application of the Iodine-Azide Reagent in Analysis of

Organophosphorus Compounds

The first report on the application o f the iodine-azide reagent for TLC detection o f phosphorothioate based herbicides (parathion, m alathion, chlorthion, m etasystox, diazinon, thiom eton) was published by Fischer and O tterback in 1959 [96], and later by Cserhati and Orsi [97], In the last two decades we have been exploring the phenom enon of induction activity, exhibited in the iodine-azide reaction by the thiophosphoryl com pounds [98-108], As the result we determ ined induction factors Fi o f the m ajor class o f organophosphorus com pounds which are presented in Table 5.

W e proposed the tentative m echanism o f induction effect exhibited by thiophosphoryl inductors [99,101], on the basis of earlier m echanistic works published by Strickland [90], M ayerstein [91] and K urzaw a [92], and concerning the reaction with the use o f thiolic inductors (Fig. 4).

(eq. I) Fi = n , / rij l3 + N, ' P S —I + HI FT Ft' \ '/

p-s-l

+ Nj"

P - S - H P S -i R' R X \ V

V - s —

so.

Table 5. induction coefficien ts o f the representative organophosphorus com pounds [98,100,102] C om p ou n d

F,“

C om p ou n d

F,u

No S tru ctu re No S tru ctu re

1 h,p o4 0 91)b [(EtO)2P(S)—S—], 450

lB b (EtO )iP(O )—OH 0 l l C b (E tS),P=S 8

211b (E tO )2P(S)—O' K+ 40 14f Ph(H2N )2P=S 142

2C b (E t 0 ) 2P (0 )—SEt 0 15AI) Ph2(EtO )P=S 105

3C1) (EtO )jP=S 6 16f Ph2(N H 2)P=S 152

6D b (E t 0 ) 2P (0 )—O—P(S)(O Et)2 6 19f P h ,P = 0 0

7D1) (E tO )2P(S)—0 —P(S)(O Et)2 12 20a M e3P=S 158

8KI> (EtO )2P ( S ) - S K+ 2 2 0 b 20c B u,P =S 133

190 b

815c (BuO )2P ( S ) - S k+ 210 b 20e Oct.,P=S 92 b

8C d (H exO )2P(S>—S' K* 137 b 201' Ph,P=S 2 1 3 b

8B e (O ctO )2P(S)—S K+ 89 b 20g M ePh2P=S 19? i»

8 C b (E tO )2P(S)—SEt 20 20h M e2PhP=S 1 9 5 b

154

J Determ ined by iodom etric titration. Determined by spectrophotometric method.

The data sum m arized in Table 5, indicate that the induction activity o f thiophosphoryl com pounds is strongly dependent on the structure, especially on the nature o f the P-S bonds. Thus, potassium diethyl phosphoroditliioate (8Bb) exhibits a high induction activity (Fi(8Bbj = 220), apparently due to the presence

o f the thiolate function in the P-S- anion. For disulfide 9Db, which may be form ally considered to be a dim er o f com pound 8Bb, the induction activity (Fj(<ji)i,) = 450) is approxim ately double that of com pound SHI), probably ow ing to the lacile cleavage of 9Db under the reaction conditions. However, the induction activity o f potassium diethyl phosphorothioate (2Bb) is only about 20% ot that of com pound 8Bb. C onversion of the thiolate function in com pound

2Bb into a stable thioester function causes a decrease in the induction activity of the resulting com pounds. Thus, com parison o f the induction activities o f a series o f triethyl thiophosphates, reveals a lack o f activities o f the phosphorothiolate 2Cb (Fj(2Cb) = 0), low activity o f triethyl phosphorothionate

(3Cb) (Fioci,) = 6) and triethyl phosphorotetrathiolate ( Jl Cb) (F i(ncb) = 8) and a slightly higher activity o f triethyl phosphorodithioate (8Cb) (F i(Sci» = 20). In contrast, phosphine sulfides 20, containing the P-S bond, exhibit rem arkably high induction effects, strongly dependent, however, on the structure. Thus, sulfides 20 with alkyl substituents exhibit induction coefficients (Fi) on the range levels 92 (Fi(2oc)) to 158 (Fi(2„a)). Phosphine sulfides with aryl substituents exhibit Fi on the level 195 (Fi(2og), F^otii) to 213 (Fi(2oi)). The replacem ent of the phenyl group in phosphine sulfide 20f by the ethoxy group leads to ethyl diphenylphosphinothionate (20f -» 15Ab) and to substantial decrease in the activity oi these com pounds (F^sai» = 105). Sim ilar replacem ent the phenyl by the am ide function affords diphenylphosphinoam idethionate (201' —> 16f) and to decrease in the activity o f this com pounds (Fi(ir,r) = 152). Surprisingly, the substitution o f the second phenyl by the antidate functions (16f —> 14f) does not cause significant change in the induction activity (Fi(i4r) = 142). Tetraethyl m onothiopyrophosphate (60b) and tetraethyl dithiopyrophosphate (7Db) also exhibit low induction activities (Fi(6Db)) = 6 and F i(7Db)) = 12, respectively).

T aking into account a high induction potency o f phosphine sulfides, and other thiophosphoryl com pounds, and the lack o f convenient m ethods o f their determ ination, the m ethod based on the iodine-azide reaction can be considered as the m ethod o f choice.

Three procedures for indirect determ ination o f thiophosphoryl com pounds based on the induced iodine consum ption were elaborated and their representative results are presented in Table 6. These include: (a) titrim etric method (on /¿mol scale) - perform ed via the induced iodine-azide reaction and the subsequent titrim etric determ ination o f the consum ed quantity o f iodine (b) coulom etric m ethod (on nmol scale) - perform ed via the induced iodine-azide reaction and the subsequent coulom etric determ ination o f the consum ed quantity o f iodine; and (c) spectrophotom etric method (on nmol scale) - perform ed via the induced iodine-azide reaction and the subsequent spectrophotom etric determ ination o f the consum ed quantity o f iodine.

Table 6. Results o f the determination o f representative thiophosphoryl com pounds - inductors [9 8 ,9 9 ,1 0 0 ,10 2 1

No Compound Titrim etric

[fimol]

Coulom ctric [nmol]

Spectrophotoinetric [nmol]

Taken Found USD

[%[ Taken Found RSD m Taken Found RSD [%] Kltl> (EtOhP(S)S' K* 1.00 1.03 8.5 0.200 0.204 2.6* 2.00 1.96 4.8 0.400 0.404 1.1* 5.00 5.06 2.7 0.500 0.520 4.6 10.00 10.00 2.4 2.00 1.97 1.4 20.00 19.70 2.0 4.00 3.98 2.9 Kite (BuO);P(S)S' K’ 1.00 1.01 7,7 0.50 0.475 8.4 5.00 4.92 3.8 2.00 2.06 3.2 20.00 19.60 2.0 4.00 3.97 2.1 Kite (OctO):P(S)S K* 2.00 2.01 3.2 6.00 6.06 2.5 10.00 10.00 0.8 9l)b [(EtO)3P(S)S-J3 0.050 0.051 2.5 0.050 0.048 8.2 0.050 0.047 7.3* 0.125 0.124 2.0 2.50 2.45 2.3 0.150 0.153 3.1* 0.375 0.370 1.6 10.0 9.90 1.8 0.250 0.245 2.4* 14 PhP(S)(NH:)i 0.2(H) 0.197 3.1 1.00 1.03 7.4 1.00 1.02 5.2 0.400 0.408 2.4 5.00 4.95 2.6 2.00 2.02 4.3 1.2 1.19 1.9 25.0 24.6 1.9 6.00 5.97 2.1 16 Ph:P(S)NHi 0.150 0.153 3.8 1.00 0.98 6.8 0.70 0.72 4.5 0.300 0.304 2.9 5.00 4.95 2.4 1.50 1.47 3.0 25.0 24.9 1.5 25.0 24.9 1.5 6.00 5.95 2.5 20a Mc.iP=S 0.150 0.146 2.8 1.00 0.98 7.1 0.50 0.52 4.2 1.00 1.01 1.6 5.00 4.96 2.3 5.0 4.92 2.4 20.0 20.2 1.7

20c Bu,P=S 0.150 0.148 3.1 1.00 0.98 7.1 0.50 0.52 3.1 1.00 1.01 1.6 10.0 9.8 2.1 5.0 4.90 1.6 25.0 25.2 2.4 20li Me2PhP=S 0.150 0.153 3.1 1.00 0.97 7.2 0.50 0.51 3.3 1.00 0.99 1.2 5.00 4.95 2.1 5.0 5.04 1.8 20.0 20.2 1.5 20« MePh,P=S 0.150 0.147 2.8 1.00 1.03 7.6 0.50 0.49 3.2 1.00 0.955 1.7 5.00 4.92 2.4 5.0 5.04 1.8 20.0 19.7 2.1 20f Ph,P=S 0.50 0.52 3.4 2.00 2.01 2.7 4.00 3.92 2.1

* U V measurements carried out in a 5 cm long cuvette.

D ifferent values o f the induction coefficients found for the same com pound depending on the method o f determ ination w ere due to different reaction conditions applied in each analytical technique. The induction coefficient has been found to be independent o f pH in the range o f 5.5 < pH < 7.5 for all determ ined sulfides. However, in the case o f phosphorothioate 2B

and/or phosphorodithioate 8B salts, as well as their disulfides 9!), induction coefficient increased with decrease o f the pH values. The use o f solutions whose pH is low er than 6 (optim al) is not recom m ended because o f the em ission o f the poisonous, volatile hydrazoic acid.

3.2. TLC Detection of Thiophosphoryl Compounds Using the Iodine-Azide Reagent

T he results o f the detection o f various thiophosphoryl com pounds by means o f UV (254 nm), the iodine detection, the m olibdate reagent and using iodine-azide reagent are sum m arized in Table 7.

Detection limits (DL) o f organophosphorus com pounds, resulting from their induction activity are strongly dependent on their structures and elem ent contributions [ 103-1051. The activity o f thiophosphoryl derivatives depends on the nature o f the P(S)„ function.

I able 7. D etection limits o f representative organophosphorus com pounds with UV detection, using iodine vapour, the m olybdate reagent and iodine-azide procedure 1105]

No Structure _{Detection limits [nmol]}

F /“ UV I2/b I rN.,'/c M olybdate“ 1254 tint] A B“ Cr D* lC b f t O - E t E t — O - P ' o - E t - _h 5 0 h _h h 3 0 ICi O 11,0-1 P h - O - P 0"^ , - 2 5 0 .5 _h J i h 5 0 lC f 0 o - P h ii/ P h -0- P v ' o - P h - 10 3 0 h _h _h 3 0 2Ck 0 P h - O — P N o -^ 2 2 2 5 0 .5 2 .5 > 5 2 5 2Ci 0 II/0 - , P h - S — P s 13 2 5 5 2 5' > 2 5 5 0 3Ci S 11,0- , P h - O — P s 0"^ 1 5 6 2 5 0 .5 2 .5 5 . 0 8Ci S 11,0 - 1 P h - S - P ' o 1 9 0 2 .0 0 .5 0 .5 > 5 1.5 8Ck s I I , s > P h -0— P x O ^ 1 6 3 2 5 0 .5 0 .5 1.5 10C1 S l l , S - i P h - O - P s - ^ 193 2 0 0 .5 0 .5 1.5 lOCk S I I , S > P h - S — P s 0" ^ 2 2 0 5 0 .5 0 .5 1.5

lI C k S

l l / S > 208 2.0 0.5 0.5 0.5 1.5

P h - S - P

Determined in solution after 15 min. 11 Brown spots on y ellow background. L White spots on y ellow back groun d.ll After pre-treatment with ammonia va p o u r.0 Blue spots. f Standard m olybdate p ro ced u re.8 M olybdate procedure (D ) with prior mineralization o f chromatographed com pounds by perchloric acid at 180 " C .h Not detectable at the level o f 50 nmol per s p o t .1 After

15 min o f exposure.

O ur results indicate that the induction activity (resulting from this detection) of thiophosphoryl com pounds is to be attributed rather to the thiolate (P 'S " ) function than to the thiol ester (P-S-R) or thiono (P=S) functions. C onsequently, phosphorodithioates and their salts, disulfides and both alkyl and aralkyl phosphine sulfides have the lowest detection limits. Triphenylphosphine sulfides and ethoxydiphenylphosphine sulfides have high detection limits - above 150 nmol presum ably ow ing to the fact, that the iodine-azide reaction is ham pered by iodide ions present in the spraying solution.

The determ inability o f organophosphorus thioam ides has been found to depend on their am ide radicals: prim ary and secondary thioam ides have low or average detection limits (0.1-2.5 nmol) whereas tertiary thioam ides remain inactive. M onothioacids, phosphorothioate and tetraethyl m onothiopyrophosphate exhibit m oderate detection limits (10-30 nmol), w hereas thiophosphate, dithiophosphate and perthiophosphate are extrem ely difficult to detect (>200 nmol). A sim ilar trend is observed for thiophosphoryl derivatives of trivalent phosphorus. D ialkylthiophosphites have m edium detection limits (20-30 nmol) this is to be explained by their isom erisation from the thiono [P(S)H], to the thiolic, [P-S-H], forms w hereas triethylthiophosphites and triethyltrithiophosphites have very high detection lim its (>100 nmol).

The iodine-azide reagent allows the m icrodetection o f aliphatic and m ixed alkyl-aryl phosphine sulfides. O rganophosphorus thioacids, their salts, partial esters and prim ary and secondary thioam ides as well as disulfides, in general, com pounds with the P=S or P-S-R functions exhibit low detection limits. All the other tested com pounds with the P=S or P-S-R functions, exhibit poor to m oderate detection limits and the application o f the iodine-azide reagent requires their prior hydrolytic activation to the P-S‘ function.

It has to be noted, that the iodine-azide test reaction applied for sulfur derivatives o f phosphorus acids may be interfered by the presence o f som e sulfur com pounds, w ithout phosphorus in the m olecule. A sim ilar interference problem also exists in the case o f m olybdate test, which is the most com m on test for phosphorus com pounds [109], Thus, the application o f the iodine-azide reaction

or the m olybdate reagent gives indiscrim inately positive results for several thiophosphoryl and sulfur derivatives. To overcom e this lack of selectivity, we additionally developed a m odified m olybdate procedure, based on the phosphoro-m olybdate reaction with prior oxidative elim ination o f potential sulfur reductants of m olybdate. As a consequence, the com bination o f the iodine-azide test with the m odified m olybdate test perm its the differentiation of thiophosphoryl, phosphoryl and sulfur com pounds.

Table 8. Detection o f stillin' com pounds and thiophosphoryl com pounds by means o f the iodine- reagent, the m olybdate reagent and the pre-oxidation-m olybdate procedure 1103,105)

Entry (No)

Compound Detection procedure"

Structure INo] Amount (l'6 Per spot) I r N,'/b Moc U ) Br2/(Ht|.) (2) Mo (1) UNO., (2) Mo (1) H2Oj (2) Mo 1 HOCH2CH2SH 1 + + — — — 10 ++ ++ — — — 2 HSC6HjC 0 2H-m 1 + — — — 10 ++ ++ + d + d + d 3 PhSSPh 1 + — — — 10 + + + d + d + d 4 PhNHC(S)NH2 1 + + + d + d — 10 ++ ++ + d + d + d 5 (EtO):P(S)ONa [2Bb] 1 +— ++ + - c + - c + _ c 10 + ++ + c + c 6 (EtO):P(S)SNa [8BbJ 1 + + + b + - c + - c to ++ ++ + c + c + c 7 [(EtO)2P(S)S]2 |9D b] 1 + + + c

+

“ Detection: ++ = strong, + = distinct, + /- = spot detectable, - = spot not detectable. ’ W hite s p o ts .c Blue s p o ts .d Brown spots with or without m olybdate spray.

T he best results are achieved when brom ine w ater is used for prelim inary oxidation. Only thiophosphoroorganic derivatives appear as blue spots in the reaction with m olybdate. Com pounds that contain only sulphur becom e visible as brow n spots.

In the light o f the results presented here, it is clear that only the iodine- azide reagent allows the selective detection of thiophosphates. O ther detection system s (UV, iodine(V), HC1(V)) routinely used in the TLC o f organophosphorus com pounds give a positive test for both phosphates and thiophosphates.

The iodine-azide reagent can be used for the detection of phosphorothioate analogues o f nucleotides in the presence o f phosphate and/or phosphonate derivatives. The com bination o f the iodine-azide and m olybdate detection procedures can be successfully applied for the chrom atographic detection and subsequent TLC differentiation o f nucleotides and their phosphorothioate analogues.

T he encouraging results on the use of the io din e-azid e reaction for TLC detection o f som e phosphorothioate derivatives of nucleotides [104] prom pted us to perform som e additional investigations on the structural factors influencing the reaction course. We focused on the induction activity o f various types of thiophosphate derivatives and the analytical repercussions, especially within the context of their selective TLC detection. In the course o f these studies, we have determ ined the induction coefficients (F,) o f several representative phosphates and phosphorothioates, in order to establish their m olecular structure-induction and detectability-induction activity relationships. T he results clearly dem onstrate the lack of induction activity exhibited by phosphates and substantial induction activity exhibited by phosphorothioates. T he latter increases with the num ber o f sulfur atoms in the inductor’s m olecule, clearly illustrating the higher induction potency o f derivatives with the thiono (P=S) rather than the thiolo (P -S -R ) functions.

The F, param eters determ ined for aqueous solutions of phosphoiothioates three times; after 5, 15 and 60 min exhibit strong time dependence, with a plateau usually reached after a reaction time o f 1 h. H ow ever, due to the m ore com plex nature o f the io dine-azide reaction during the TLC detection o f phosphorothioates, the correlation betw een detection limits and induction coefficients is better for shorter periods o f exposure time (1 to 5 min). The results illustrate the reasonably good correlation betw een the induction potency (F, ) o f phosphates and phosphorothioates and their detectability.

Fj(5') = 9 (DL = 25 nmol) F|(5') = 141 (DL ” 2,5 nmol) Fi(5')=199 (DL = 0,5 nmol)

Phosphates exhibit no induction potency and are not detectable on TLC plates. Sugar phosphorothioates (Fig. 2) exhibit even more specific induction properties. T heir induction activity is dependent on their m olecular structure, and increases strongly with reaction time [105]. Thus, the deoxyglucose phosphorothioate Q i)-S -P has m oderate induction activity (Fi(y) = 47), glucose derivatives have m edium induction activity (90 < Fi(y) < 107), and the com pound with the C (3rS-P linkage pattern exhibits the highest induction activity (F,(5') = 280) o f the series. These results suggest that S- alkylphosphoro(thio)ates with the C (i)-S -P linkage pattern exhibit lower induction activities than those with other [e.g. at C(3)] alignm ents of phosphorothioate in the sugar ring. The deoxysugar derivative has lower induction potency than the glucose phosphorothioate. The induction activity o f all sugar phosphorothioates exhibits a strong dependence on the reaction time as the P-S-C bond breaks. T he induction coefficient ( F , ) after 60 min is five times higher than after 5 minutes.

The correlation between DL and F, appears to be yet m ore com plex in the case o f sugar derivatives than in the case of the series o f phosphorothioates. Surprisingly, the high induction potency that these com pounds exhibit in solution is not reflected in their TLC detection on silica gel plates. Prolongation ot the exposure tim e does not influence the detectability. This may be due to the fact that the m echanism o f the iodine-azide induced reaction is m ore com plex on silica gel plates.

The relatively poor detection o f certain phosphorothioates in the iodine- azide m ethod can be greatly enhanced by hydrolytic pre-treatm ent o f the chrom atographic plate. Such a pre-treatm ent step m ost probably involves the hydrolytic splitting o f the P -S - C (sugar) bonds and can occur, depending on the structure, either under basic or acidic conditions. Thus, com pounds with a phosphorothioate m oiety at the C (1) carbon o f the sugar unit [the C (i>-S-P linkage], are difficult to detect using the iodine-azide reagent (D L = 100 nmol). Their detection rate increases strongly if the plate is pre-exposed to am m onia vapour (DL = 3 nmol) or HC1 vapour (DL = 2 nmol). Such high increase in the detectability suggests, that during the hydrolytic pre-treatm ent o f sugars

thioloesters corresponding m ercaptosugars are form ed, which are more potent inductors in the io d in e-azid e reaction.

Fig. 6. H ypothetical course o f the digestion o f 1-thiophosphorylsugars, occurring during the treatment with HCI or am m onia vapour.

Phosphorothioates with a thiophosphoryl function at the Cp> atom o f the sugar unit [the C p j-S - P linkageJ have been found to have substantially higher detectability. T he iodine-azide method allows detect these com pounds at the level of 20 nmol per spot. M oreover, when alkaline hydrolysis or acidic hydrolysis is used the detection level increases to 2 nmol and to 0.5 nmol respectively.

The results o f som e other representative procedures applied for the TLC detection o f phosphates and phosphorothioates, are sum m arized below.

All tested com pounds give a positive test reaction when exposed to iodine vapour (brow n spots on a yellow background) and their detection limits range betw een 0.2 to 0.5 nmol per spot for com pounds thiophosphates and at 2 .5 -1 0 .0 nmol per spot for sugar derivatives. UV detection (at 254 nm) leads to am biguous results. Thus, the phosphorothioates with an arom atic ring and/or the phosphorothiono (P=S) moiety exhibit detection limits at the level 2.0 to 25 nmol per spot, w hereas phosphorothioates with aliphatic substituents are only poorly detected, with DL below 100 nmol per spot. The detection o f phosphates and/or phosphorothioates using m olybdate reagents depends on the stability of their phosphoester or phosphothioester functions. In the case o f application o f a m ilder procedure A - w ithout prior hydrolysis with perchloric acid - the DLS vary from 1.5-5.0 nmol for com pounds with the P=S linkage, and 2 5 -5 0 nmol for com pounds with the P-S-C linkage [104], In the sugar series, the com pound bearing with the Cyj—S—P linkage has a DL o f 0.5 nmol, w hereas com pounds with the C (1)- S - P linkage are very difficult to detect (D L S > 100 nmol per spot). These detectabilities substantially increase when chrom atographed com pounds have been subjected to prior hydrolysis with perchloric acid and the plate has been heated to 180°C (procedure B) [103,109,110],

The application o f the iodine-azide procedure, com bined with the m olybdate procedure, for the selective TLC analysis o f phosphates and thiophosphates m ixtures (entries 1-3) is illustrated in T able 9.

OH OH

T able 9. TLC analysis o f the mixtures o f phosphates and phosphorothioates 1105] Entry (N o) M ixture o f com pounds “ Detection system b UV /R Ke I j d /R hc i2-n3-/c / I V M olibdate r A ‘/R hc B " /I V 1 Q II, P h -O —P, and S

t

Taken 10 jig o f each com pound. b++ = strongly detected, + = distinct detection, - = not detectable. c S ilica gel/benzene-ethyl acetate (9 : 1; v/v). d Brown spots on y ello w background. c W hite spots on y ello w background. 1 Blue spots. g M olybdate reagent; procedure A. h M olybdate reagent; procedure B.

Thus, the cyclic phosphates were chosen for our research so as to differentiate hydrolysis susceptibility. We chose three m ixtures containing phosphates and tw o with sulfur atoms in different positions. As we had expected, only thiophosphates gave positive tests with the iodine-azide reagent. Phosphates were not visible also when the m olybdate reagent without prior hydrolysis was applied (procedure A). The m olybdate used in the more drastic procedure B was unselective and all derivatives appeared as blue spots.

3.2.1. TLC Detection of Phosphorothioate Analogues of Nucleotides

The group o f thiophosphoryl com pounds o f increasing im portance both in pure and applied chem istry, includes phosphorothioate analogues of nucleotides and oligonucleotides presented by the general structure given on Fig. 3. Som e representative structures are presented on Fig. 7.

0TP(T*=> DMT(T)ODMP DMT(T)DTP

Fig. 7. Representative structures o f thiophosphate nucleosides applied.

Despite the fact that all nucleosides and their derivatives are easily detectable by UV spectroscopy it was crucial to find a m ethod for the selective identilication ot phosphorothioate analogues of nucleotide analogues in m ixtures containing also “non-sulfurized” nucleic acid com ponents.

Table 10. D etection lim its o f phosphorothioate analogues o f nucleotides with UV detection (254 nm), using iodine (vapour), the molibdate reagent and the iodine-azide detection reagent [104]

Entry (No)

Compound Detection system

(nmol per spot)

TLC [RfI

Structure * _{h "} UV h-N j'/c Molybdate'11

Her

1 PhO-OTP 0.1 1.5 0.3 1.5d .8 0.73"

2 5 ’-CM P 2.0 6.0 - 2.0d 0.37'

4 D M T(SlU)OTP 0.3 0.6 1.2 0.1"; 10ru 0.1 0 .5 0 k 5 DM T(sjAB/')OTP 0.3 0.6 1.0 ().lc 0.1 0.73' 6 DM T(T)O TP 0.15 0.6 0.5 0.1° 0.1 0.69' 7 D M T (dA l>y“)OTP 0.5 0.6 1.3 0.4° 0.3 0.52' 8 O TP(AcT) 0.2 1.2 0.6 12d j 0.69' 9 OTP(dAcG'Br) 0.2 0.2 0.6 2 .0d j 0.60' 10 D M T(T)D TP 0.15 0.6 0.5 0 .1 5C; l()u 0.15 0.67' 11 D M T (dA B/)DTP 0.15 0.6 0.5 0 .1 5‘ 0.15 0.64'

“Abbreviations: A, G, U, T: adenosine, guanosine, uridine, thymidine; siA, siG, siU; corresponding ribonucleosides blocked at 2' position with 'BuM eiSi group; dA, dG: corresponding deoxyribonucleosides; Ac: acetyl; iBr: isobutyryl; Ph: phenyl; Bz: benzoyl; Pya: N- m ethylpyrrolidin-2-ylidenyI; DM T dimethoxytrityl; OTP: 2-thio-l,3,2-oxathiaphosph olan e; DTP: 2-thio-l,3,2-dithiaph ospholan e; ODPP: diphenylphos-phate (detailed structures o f representative com pounds are given in Fig. 7). b Brown spots on y ellow background. c W hite spots on brown background. d Blue spots on buff background.c Pink spots. ' After treatment with a TFA solution (10% ) in MeOH and redevelopm ent with appropriate solvent system . “ Not detectable up to level ol 50 /ig per spot. h S ilica gel/M eC N . ' S ilica g e l/M eO H .J S ilica gel/benzene-M eO H ( 9 : 1) . k A pink spot containing DM T cation was shifted up. 'C ellulose/M eO H -form ic acid -H 20 (80:15:5).

In the light o f the results presented in Table 10, only the iodine-azide detection reagent allow s the selective detection o f the thiophosphate (phosphorothioate) system s. O ther applied detection system s, routinely used for the TLC o f organophosphorus com pounds (UV, iodine, HCl(v), the m olibdate reagent) gave a positive test for both phosphates and thiophosphates.

In contrast, the iodine-azide reagent detects sulfur-containing m olecules and therefore can be useful for the detection o f phosphorothioate analogues of nucleotides in the presence o f the phosphate and/or phosphonate type derivatives. As a consequence, the com bination o f the iodine-azide detection procedure and the m olybdate detection procedure can be applied for the chrom atographic detection and subsequent TLC differentiation o f nucleotides and their phosphorothioate analogues (Table 11).

As stated above, detection limits o f thiophosphoryl com pounds correspond to their induction potency in R aschig’s reaction which in turn depends on the inductor’s structure. Therefore it was o f great interest to exam ine the structure-induction activity relationship for more com plex com pounds namely phosphorothioate nucleotide analogs. N ucleotide derivatives containing 2 -thio-l,3,2-oxathiapho sphalane (OTP) or a 2 -thio -l,3 ,2-dithiap ho sph alan e (D TP) ring w ere analysed using the iodine-azide reagent [ 104J.

Table 1 1. TLC analysis o f the mixtures o f nucleotides and their thiophosphoryl analogues [104] E ntry M ixtu re o f D etection sy stem 11 [D etection/(R p)h] (N o) c o m p o u n d s " UV [254 nm ]

lodinC(v) M olyb d ate I r N /

A B 1 PhO-OP and PhO-TOP and PhO-TDTP +(0.35) +/(0.46) +/(0.51) ++(0.35) ++/(0.46) ++/(0.51) +/(0.46) ++/(0.5I) ++(0.35) ++/(0.46) ++/(0.5I) ++/(0.46) ++/(0.51) 2 ODPP and PhO-TOTP and PhO-TDTP ++/(0.41) +/(0.51) ++/(0.58) +/(0.41) ++/(0.51) ++/(0.58) -/(0.4I) ++/(0.51) ++/(0.58) +/(0.41) ++/(0.51) ++/(0.58) ++/(0.41) ++/(0.51) ++/(0.58) 3 T (D TP) and D M T(T)O D M P ++/(0.13) ++ /(0.20) + + /(0 .13) ++ /(0.20) ++d/ ( 0 . 13) ++7(0.20) ++7(0.13) ++7(0.20) ++7(0.71) ++(0,13) _ e 4 O TP(TA c) and DM T(T)DTP ++/(0.17) ++/(0.37) ++/(0.17) ++/(0.37) ++d/(0.17) ++7(0.37) ++7(0.17) ++7(0.37) ++7(0.76) ++(0.17) ++(0.37)

‘‘Taken amount: 10 nm oles o f each nucleotide per spot (abbreviations as in Table 1 0 ) .b Detection: ++ = strong, + = distinct, +/- = spot detectable, - = spot not d etecta b le.c Pink spots, d Blue spots on bu ff background. 1 After treatment with TFA and re-developm ent with an appropriate solvent system . 1 W hite spots on y ello w background. 8 Taken at amount 50 /tg per spot. h S ilica gel: benzene-ethyl acetate (9:1) (line land 2), benzene-M eOH (9:1) (line 3 and 4).

Iodine detection o f phosphorothioates is more sensitive than that exhibited for their phosphate analogs. Phosphorothioates with arom atic system are visible in UV light. Iodine-azide detection o f phosphorothioates also proves to be a highly sensitive m ethod (0.15 to 1.3 nmol per spot) and the detection sensitivity, as in the case o f sim ple derivatives, depends on the structure o f the m olecule. O-aryl phosphorothioates are detectable at the level o f ca. 0.3 nmol. The substitution o f sulfur by selenium atom does not affect the DL value. The detection limits for phosphorodithioate nucleotide analogues are in the range o f

0 .5 -1 .3 nmol. No significant differences are observed in the detection of phosphorothioate nucleotides with various nucleo-bases and only small differences betw een the series o f ribonucleotides and deoxyribonucleotides. M ore distinct differences are observable in the series o f nucleotides with phosphorothioate functions attached at the 2 ’ and 5 ’ positions o f the sugar ring o f nucleosides. The detectability o f phosphorothioate nucleotide analogues tends to increase with the increase o f sulfur content in the phosphorothioate function and in the case o f phosphorotrithioate D M T(dG )D TP the detection level even reaches the level o f 0.15 nmol per spot.

The iodine-azide reagent does not exhibit any activity tow ards phosphate com pounds which do not contain sulfur such as DM T(T)O D PP, DM T(T)O D M P, (see Fig. 7) cytidine 5 ’-m onophosphate and guanosine 5 ’- m onophosphate. T hese com pounds cannot be detected even at the level of 100 nmol per spot.

The detection o f phosphorothioate nucleotide analogues by m eans o f the m olybdate procedure leads to am biguous results as both phosphorothioates and phosphates react with the m olybdate reagent form ing blue spots on a white background 1110], However, since this procedure requires strong acid m edium, the dim ethoxytrityl protected nucleotides (D M T) appear on chrom atographic plates as pink spots which is due to dim ethoxytrityl cation form ation. T he strong absorbance o f dim ethoxytrityl cation at ca. 500 nm sufficiently masks characteristic blue spots that result from the reaction o f the m olybdate reagent with the phosphates and phosphorothioates. Therefore, the application o f the m olybdate procedure for the TLC detection o f D M T-nucleotides requires some m odification.

The developed chrom atographic plate has to be sprayed with a 10% solution o f trifluoroacetic acid then it has to be redeveloped before the m olybdate agent is used. D etritylated nucleotides appear as blue spots and DM T-TFA derivatives appear above as pink spots.

3.2.2. TLC Detection of Sulfur-Containing Aminophosphonic Acids

The detection lim its (DL) for cysteines (Cys and C ys1’), hom ocysteines (H cys and H cysp) and m ethionines (M et and M et1’), by m eans o f the iodine-azide reagent and the other representative detection procedures are sum m arized in Table 12.

The results o f the reaction o f the exam ined am ino acids with the iodine- azide reagent, carried out both in solution and on a TLC plate, generally presented poor correlation.

Thus, C ys (Fi = 325), C ysp (Fj = 138) and H cys1’ (Fj = 60) exhibit a DL = 30 nmol, w hereas H cys (Fj = 95) is detectable at DL = 30 nm ol per spot. Both

m ethionines (M et and M et1) have not been characterized hy induction coefficients due to their reaction with iodine alone, occurring in a solution [1 0 1 1. However, their treatm ent with the iodine-azide reagent on T LC plates gave typical positive test results by formation o f characteristic white spots on a yellow background with the DL = ca. 20 nmol per spot.

All exam ined am inophosphonic acids, as well as the thiolic am ino acids, can be detected by means o f the m olybdate detection reagent. T hus, for M et1’ the detection limit was as the order as 5 nmol, and for C y s1’ and H cys1’o f the order of 20 nmol. Surprisingly, for Cys and Hcys (am ino acids without phosphonic m oiety) the detection limits was 30 nmol, whereas M et and Gly are not detectable up to a level o f 50 nmol per spot. This phenom enon can be explained by the interaction o f the sulfydryl groups o f these am ino acids with the m olibdate reagent. T he application o f the iodine-azide detection allows visualization o f the sulfur-containing am ino acids on TLC plates in mixtures with other am ino acids. The application o f the pre-oxidation step prior to the m olibdate detection allow on the differentiation o f C y s1’, H cys1’ and M et1’ from their carboxylic analogues (Cys, Hcys and Met) [107],

Table 12. Comparison o f the TLC detection properties o f sulfur-containing amino acids and their am inophosphonic analogues 1107]

A m in o acid s _F, T L C D etectio n L im its

[n m ol per sp ot] R .“ A b brcv. S tru ctu re UV I2b I r N 3-/c N in h .d M o' C ys H S - C - C — H* n h2 o h 325 i 20 20 2 0 30' 0.19 H cys 0 H S - [ C H 2]— C - Ą n h2 o h 95 i 5 30 5 3 0 1 0.45 Met 0 M e - S —[CH2]— C — ^ n h2 o h h i 5 20 20 _ j 0.51 C y sp 0 H II^OH h-s- c- c- pC H? 1 OH 2 n h2 138 i 20 20 2 0 20 0.25

H cysp O H II .OH h - s - [ Ch2,— c - p C qh nh2 60 i 5 30 5 30 0.40 M etp M e-S-[CH 2]— C—P C °H 22 1 OH n h2 _h i 5 20 1 5 0.46

Induction factor of am ino acids (F|) determined in solution. h Iodine procedure: brown spots on yellow background. c Iodine-azide procedure: white spot on y ellow back-ground. d Ninhydrine procedure: blue spots. c M olybdate procedure: blue spots. 1 Spots appeared after 15 min o f exposition time. STLC system : cellu lose plates DC vs. n -B u O H -A c O H -w a ter (12 : 3 : 5). h D oes not induce the iodine-azide reaction in so lu tio n .' Not detectable in UV up to the level o f 100 nmol o f amino acid. J Not detectable up to the level o f 50 nmol o f amino acid.

3.2.3. Unusual Induction in the Iodine-Azide Induced Reaction

O ur further research on the application o f the iodine-azide reagent for organophosphorus com pounds analysis has shown that non-sulfur com pounds induce the iodine-azide reaction [106], The results supporting this thesis are presented in Table 13.

I able 13. Comparison o f the I LC detection lim its (/zg per spot) o f phosphorus and otganophosphorus com pounds by means o f UV (254 nm), iodine vapour, the iodine-azide reagent other representative reagents [106]

Compound _{Detection Limits (n g per spot) “}

R,

No Structure UV _{I2b M n ' Mo B Ninh."}

1 ( H 0 ) 3P = 0 - — — _{l f 0.2 10 0.51 m} lC b (E t 0 ) 3P = 0 - 10 10 b — — — _{0.53 "} lC f (PhO)3P=0 3 10 3 b — — — _{0.68 11} 12 _{l? „ 0 - H} h- pC O—H 10 3m l f 10 20 0.66 m 12Ba ( M e 0 ) 2P (0 )H - 10 3 3 — _{10 0.48 n} 12Bb ( E t0 )2P (0 )H - 10 10 3 — _{10 0.55 "} 12Cf (P h 0 )2P (0 )H 10 10 10 3 3 10 0.70" 12Ca (M eO )3P - 3 10 3 3 10 0 .50" 12Cb (EtO)jP - 3 10 3 3 10 0.55" 12Cf (PhO)jP 10 10 3 3 30 1 1 0.72 "

12b O iL o- h Et— PC O - H l f 1 2 0k 0.72 12n X X 1 o o \ / 0 = 0 - 1 X X O -O 1 U J 1 r 10 20 k 0.64 12r _H 0_{I l ^ o - H} Et— C -pC T O - H n h2 l r 0.2 0.1 1 0.30 m 21 _{0 X} 1 0 = 0 . / \ I X 10 30 d'b 1 10 30 0.36 m 22(1) PhiP—OEt 10 10 I 0 b 3 3 3 0.51 " 23f PhjP 1 1 3 3 3 1 0.71 "

“ = not detectable at 50 |ig per spot. b Brown spots. L' W hite spots on y ello w background. d Appear after 15 m in .c Light green-grey spots on light-brownish background. rPink spots without heating or after preheating to 100 °C . g Blue spots - after preheating to 100 °C or after irradiation at 3 6 0 n m .h Pink spots after preheating to ca. 1 0 0 °C .1 Pink spots without h e a tin g .k W hite spots on faint-pink background. m n TLC system s: (ra)cellulose/iB uO H -T H F-w ater-acetone-T os-O H (8 ml : 6:5 ml : 1 ml : 0.3 g); ‘"'silica gel/acetone.

However, until now, there have not been any reports in the chem ical literature describing the induction o f the iodine-azid e reaction caused by com pounds which do not contain sulfur.

T he results o f the TLC detection of the broad spectrum o f tri- and pentavalent phosphorus com pounds illustrate the possibility o f differentiation o f these com pounds. The m ajority o f di- and trialkyl phosphonic acid esters [(RO )2PHO and (R O ^P ] as well as triphenyl phosphine give a positive reaction with the iodine-azide reagent. O ther derivatives of trivalent phosphorus with P-C bonds appear as brow n spots. Phosphonic and phosphoric acids are inactive. The m olybdate reagent unlike the iodine-azide reagent does not allow to differentiate betw een tri- and pentavalent phosphorus com pounds.

Such unexpectedly good detection o f trivalent phosphorus com pounds, which is com parable to that o f the sulfur com pounds, can be explained by their reactivity. D etection sensitivity depends both on the structure o f the phosphorus com pounds and on the type of IL C plates used. Thus, triphenyl phosphine is detected as white spots on silica oxide plates, or brow n spots on alum ina oxide or on reversed-phase plates. However, in spite o f the distinct induction activity o f these phosphoryl derivatives on the TLC plates, they are totally inactive in R aschig’s reactions carried out in solution. The determ ination o f the induction