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Pyrolysis Mass Spectrometry of Protected Phosphotriester Oligodeoxyribonucleotides

J. Ulrich M. J. Bobenrieth Laboratoire de Spectrometrie de Masse, DCh/DCA/CAG/EAPC, Centre d'Etudes Nucleaires de Grenoble, 85 X, 38041 Grenoble Cedex, France

R. Derbyshire, F. Finas, A. Guy, F. Odin, M. Polverelli, and R. Téoule* Laboratoire de Radiobiochimie, Départment de Recherche Fondamentale, Centre d'Etudes Nucléaires de Grenoble, 85 X , 38041 Grenoble Cedex, France Z. Naturforsch. 35b, 212-216 (1980); received July 16, 1979 Phosphotriester Protected Oligodeoxynucleotide, Pyrolysis Mass Spectrometry

Pyrolysis mass spectrometry is shown to be a useful tool in the analysis of the protected intermediate deoxy oligonucleotides synthesized by the phosphotriester approach. Characteristic ions of the protected nucleic acid bases and terminal substituents appear in the mass fragmentation pattern which allows a rapid control of the attachment of the polynucleotide block in the chemical synthesis.

Under pyrolytic conditions DNA has been subjected to mass spectrometry analysis [1-3]. Cleavage of the phosphodiester bonds linking the nucleotide residues followed by subsequent frag-mantation and rearrangement result in ions characteristic of the nucleobases and deoxyribose fragments. The method has been extended to di-nucleotides protected on the amino and hydroxyl functions which have also an internucleotidic bond of the phosphodiester type [4].

Independently [5], we have studied the pyrolysis mass spectrometry fragmentation of fully protected oligonucleotides obtained as key intermediates in the phosphotriester approach of gene synthesis. We wish to report herein the results obtained with these phosphotriester products. The method enables the direct analysis of very small amounts of material without prior chemical derivatization or enzymatic treatment.

Experimental Synthesis of the fully protected phosphotriester oligonucleotides

The hexanucleotide d[MeOTr - bzA(ClPh) -bzA(ClPh) - acG(ClPh) - bzC(ClPh) - T(ClPh) - Tac) was prepared by condensation of d [MeOTr -bzA(ClPh) - bzA(ClPh) - acG(ClPh, CNEt)] and d[bzC(ClPh) - T(ClPh) - Tac] by the general proce-

* Reprint requests to Prof. R. Teoule. 0340-5087/80/0200-0212/$ 01.00/0

dures previously described for phosphotriester oligonucleotide synthesis [6-17].

The protected trinucleotides were obtained by the method recently described by Cashion et al. [6]. d[MeOTr - bzC] was phosphorylated and then condensed with dThd to give d[MeOTr bzC(ClPh) -T]. The same procedure was used to prepare d[MeOTr - bzC(ClPh) - T(ClPh) - T]. d[MeOTr -bzC(ClPh) - T] was phosphorylated by an excess of imidazolyl-triazolyl-p-chlorophenylphosphate and the reacted material condensed with dThd. Acetyla-tion and detritylation were performed as usual [10]. A similar method was used to obtain d[MeOTr -bzA(ClPh) - bzA(ClPh) - acG], which was sub-sequently phosphorylated and cyanoethylated as described previously [10, 11].

The Cashion procedure [6] is simple and efficient at least for the synthesis of di- and trinucleotides. For higher oligonucleotides the yields tend to decrease. Under the set of conditions used by the authors (for example d[MeOTr - bzC] 1 equiv., P-CIPI1PO2CI21. 2 equiv., triazole 4 equiv., triethyl-amine 4 equiv., CH3-imidazole 16 equiv., dThd 3 equiv.) it appears that 3 ' ->3 ' condensation, as shown by 31P NMR (unpublished work), is very small and that the corresponding products are eliminated during the chromatographic purification steps.

The products were separated by preparative high pressure liquid chromatography using silica gel fine particles thus improving the practical yields of final products compared to those obtained by thin layer silica gel chromatography. Formation of highly coloured side products, which proved troublesome in the separation step, has since been reduced by replacing pyridine by dioxane or tetrahydrofuran as the condensation solvent. However, traces of pyridine seem to be useful as catalyst.

J. Ulrich et al. • Pyrolysis Mass Spectrometry 213

Characterization of the product The hexanucleotide was detritylated by a mixture

of benzene sulfonic acid in chloroform-methanol [10]. The phosphate and amino protecting groups were eliminated by treatment with ammonia [10]. The deprotected hexanucleotide was purified by PARTISIL 10 SAX High Performance Liquid Chromatography [18]. The sequence was verified by the method of van Boom using phosphodiesterase [19].

Mass Spectrometry Analysis Mass spectra recording

The mass spectrometer, AEI-Kratos, was con-nected to a computer system (Sysmas-Tele-mecanique). Samples (1-2 jug) were placed at the end of a glass tube and introduced into the ion-source, heated at about 180 °C, working in electron impact at 70 eV.

At fixed intervals a mass spectrum was scanned and memorised. If necessary, high resolution spectra were scanned with perfluorokerosene to give the atomic composition of each ion. Reproducibility of spectra was controlled.

Substituents After ionization, the 5'-terminal substituent

paramethoxytrityl radical is liberated thermally and appears at m/e 273 with relative fragments (m/e 165 by MeOC6H5 elimination). The trityl radical may be hydrogenated in the ion source or in the sample and then appears at mje 274 (MeOTr H)+- and m/e 197 (phenyl elimination). The molecule MeOTr OH (molecular ion mje 290 and main fragment at m/e 213, phenyl elimination) is of a lower intensity.

Ions corresponding to MeOTr OH with one or two deoxyribose residues appeared at:

m/e 370 for [MeOTr OH + 80]+-, m/e 450 for [MeOTr OH + 2 x 80]+\

When the 3'-terminal phosphate was cyano-ethylated the acrylonitrile molecular ion at m/e 53 was observed: CH2 = CH-CN.

The molecular ion derived from product 9 (Fig. 1) is also detected and is of relatively high intensity when the nucleotide is unsubstituted in the 5' position.

When the 3'-hydroxyl terminus is acetylated acetic acid is liberated thermally and observed at m/e 60 and 43.

BASES AND NUCLEOSIDES

B H = bz Ade

ac Qua

Thy

bz Cyt

SUBSTITUENTS

•C t V *

Co) Ö

OH I o—p=o I OH

6

CH, = C H CN

V C M ( 0 ) - o - f = o

OH 7

OH

OCH2CHJ CN

S » Fig. 1. Suggested formula for the products obtained through pyrolysis.

Application to the control of synthesis Comparison of the fragmentation pattern of the

hexanucleotide and of the two trinucleotides used in its synthesis is shown in Fig. 2. In this way, it is possible to follow the progression of the synthesis.

However, it is important to note that the plot of intensity against time for a given ion is dependent on the nature of the ion and of the oligonucleotide from which it is derived. Fig. 3 shows the intensity of the main molecular ions against time during the pyrolysis of the hexanucleotide.

Characteristic ions

Pyrolysis of the fully protected deoxyribo-nucleotides in the mass spectrometer ion source gave rise to volatile products which were identified by their mass fragmentation pattern (Fig. 1).

Nucleobases Benzoyladenine (m/e 239) gave m/e 211 and 210

by carbon monoxide and hydrogen elimination and m/e 105 (C6H5CO)+.

Acetylguanine (m/e 193) gave m/e 151, deprotected guanine, by ketene elimination.

214 J. Ulrich et al. • Pyrolysis Mass Spectrometry 214

d f M e O T r . b z A _ ( C l C , H 4 ) _ b z A _ ( C I C , H 4 ) . a c G t C I C , H 4 l C N E t > ]

1 1 2 8

x 1 0

Ji lMl|lMi|nii[iiii|ilii|nii|llil|lhi|ilii[ilii|iiii|iili|iili|llh|ilii|i

A A

2 3 9

lil|iiii|iiil|iiil|lln|ii'

G A A G M T r

2 7 3 2 9 0 3 1 9 3 5 3

|l IM |MI I jl III |1 llljHMj| ||| || Hl j| || ||| || Ijhl l|| II l|l lltj

250 300 YmPTp

350 ll|lllljllll|llll|llll|llll|IIH|lll

400

loo 128

126 /

d [ M e O T r . b z A . (Cl C , H 4 ) _ b z A ( C I C , H 4 ) . a c G ( C I C , H 4 ) . b z C (C l C , H 4 ) _ T _ (C l C ^ J - T O A c ]

C T A C A

2 0 6 2 1 5 2 3 9

i ttiltrVr

x10

G A C A M T r

2 7 3 2 9 0 3 1 9

2 9 5 I J

/ | "I" l"'T" ,mi llil|lin|iill|iiii|

300

3 5 3 3 7 5 4 1 4

ll|llll|llll|llll[MII|llW|llll|llll|llll|llll|llll|IMI[llll|ini|llll|llll|llll|ll<|'

Fig. 2. Comparison of mass spectra. The hexanucleotide is obtained by condensation of the two trinucleotides. The characteristic ions are indicated by A: adenine, G: guanine, C: cytosine, T: thymine, MTr: methoxytrityl.

J. Ulrich et al. • Pyrolysis Mass Spectrometry 215

acG(ClPh) - bzC(ClPh) - T(ClPh) - Tac].

Benzoylcytosine (m/e 215) gave m/e 214 (H elimi-nation) m/e 187, 186 by carbon monoxide and hydrogen elimination, m/e 138 by phenyl elimination and m/e 105 (C6H5CO)+.

Thymine (m/e 126) gave m/e 83 by cyanic acid elimination.

In addition each nucleobase was observed asso-ciated with one or two deoxyribose residues, these ions being obtained probably by monochlorophenyl-phosphoric acid 6 elimination. These molecules are designated by [BH + 80] and [BH + 2 x 8 0 ] , the latter being of low abundance.

Their molecular ions are:

m/e 319 and 399 for benzoyladenine, m/e 295 and 375 for benzoylcytosine, m/e 273 and 353 for acetylguanine, m/e 206 for thymine (m/e 286 was not ob-served).

Parachlorophenylphosphoric acid was identified though its molecular ion (m/e 208, 210) is weak, being of low stability (its main fragment ion, para-chlorophenol, (m/e 128, 130) is derived mainly from pyrolysis molecules).

Thymine, whose simple composite ions ( B H + 8 0 •••) were of low intensity, gave rise to an ion of higher m/e value 7 ( C i e H ^ O v P C l ) , which is a (BH + 80) ion with a p-chlorophenylphosphoric acid m/e 414 attached. It appears at the end of pyrolysis.

The methoxytrityl ions can be detected just after sample introduction into the ion source and are the first ions to be liberated. The chlorophenyl ions attain a maximum of intensity later in the pyrolysis.

Nucleobase ions also show different rates of evoluation. Thymine ions are relatively low in reaching a maximum in intensity and the ions [Thy + 80]+' are of lower relative abundance than the other [BH + 80]+- ions.

Conclusion Due to the high molecular weights and the

complexity of protected phosphotriester oligo-nucleotides, which are isolated in small quantities as a mixture of diastereoisomers, the current available spectroscopic methods, NMR, IR, conventionnal mass spectrometry, are unsuitable for their charac-terization. Usually they are neglected and the protected phosphotriester oligonucleotides are not characterized. The structures of the resulting oligo-

216 J. Ulrich et al. • Pyrolysis Mass Spectrometry 216

nucleotides are thus determined after deprotection by the "fingerprint" method [20] or by the system of Maxam and Gilbert [21]. The structures of the protected oligonucleotides are inferred from the results obtained after their deprotection. However, it is useful to have a method to check rapidly the nature of the protected oligonucleotide after the condensation step in the phosphotriester approach.

The method has proved equally useful with oligo-nucleotides having the more currently accepted protecting groups: dimethoxytrityl, anisoyl and isobutyryl.

This work was supported by grant No. 017 ATP 52.77.84 from the Institute National de la Sant6 et de la Recherche Medicale (France).

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