Journal of Chromatography, 389 (1987) 165-176
`Elsevier Science Publishers B.V., Amsterdam — Printed in The Netherlands
`
`CHROM. 19 176
`
`COLUMN CHROMATOGRAPHIC PURIFICATION OF GUANYLATE-RICH
`SYNTHETIC OLIGODEOXYRIBONUCLEOTIDES
`
`HERBERT SCHOTT*, ROLF SEMMLER and HEINER ECKSTEIN
`Institut fiir Organische Chemie, Universitat Tiibingen, Auf der Morgenstelle 18, D-7400 Tiibingen-1
`(FLR.G.)
`(Received October 20th, 1986)
`
`SUMMARY
`
`The purification of the guanylate-rich DNA fragments d(T4G4), d(G4T4),
`d(G4T4G4), d(T4G4T,4) and d(T,G,T4G,) using column chromatography ona pre-
`parative scale is described. The crude oligonucleotides were obtained after deprotec-
`tion of the chemically synthesized compounds. The separation can be performed with
`commonly used sorbents (DEAE-cellulose, QAE-Sephadex, Nucleosil C,,. Partisil
`10-SAX), however with high losses during the chromatography. Guanylate-rich oli-
`gonucleotides of different chain lengths associate with each other, thus causing iden-
`tical compounds to be contained within different peaks. At the same time, part of
`the product remains irreversibly adsorbed on the sorbent. The recoveries could be
`improved by application of ion-pair reversed-phase high-performance liquid chro-
`matography. Theoligonucleotides were fractionated with linear increasing gradients
`using acetonitrile as the organic modifier and tetrabutylammonium hydrogensulphate
`as the ion-pair reagent.
`
`INTRODUCTION
`
`The synthesis of guanylate-rich DNA fragments is much more laborious than
`the synthesis of comparable oligonucleotides, which contain only few or no guanylate
`monomer units in their sequences, Besides distinctly lower yields obtained in the
`condensation reaction, there are additional difficulties in separation, isolation and
`identification, which have not been solved.
`We have synthesized the guanylate-rich oligonucleotides d(T4Ga4), d(G4T4),
`d(G4T4G,q4), d(T4G4T4) and d(T4G4T4G,4) in preparative amounts. These oligonu-
`cleotides correspond to fragments of the terminus of the macronuclear DNA of hy-
`potrichous ciliates'~°. The syntheses were carricd out in solution according to the
`phosphotriester method and will be published elsewhere’. The same oligonucleotides
`were preparedin three ways, applying differently protected guanylate monomerunits.
`The results of thirty different condensation reactions, several of which have been
`performed repeatedly, may be summarized as follows: the synthesis and isolation of
`the protected guanylate-rich oligonucleotides can be achieved in gram amounts. The
`
`0021-9673/87/$03.50
`
`© 1987 Elsevier Science Publishers B.V.
`
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`166
`
`H. SCHOTT, R. SEMMLER, H. ECKSTEIN
`
`condensation reactions result in good yields, which are essentially independent of the
`kind of protecting groups and of the choice of the agents used for the condensation.
`However, serious difficulties arise during the chromatographic purification of the
`deblocked oligonucleotides as will be reported in this paper.
`
`EXPERIMENTAL
`
`Materials
`Sephadex G-15- and QAE-Sephadex A-25 were obtained from Pharmacia
`(Uppsala, Sweden), DEAE-cellulose from W. R. Balstone (Maidstone, U.K.), Dowex
`50W-X8 from Serva (Heidelberg, F.R.G.) and Nucleosil 7 C,g from Macherey &
`Nagel (Diiren, F.R.G.). Tetrabutylammonium hydrogensulphate (TBA)for ion-pair
`chromatography was from Merck (Darmstadt, F.R.G.).
`
`Fractionation
`Column chromatography of the deprotected oligonucleotides. The deprotected
`oligonucleotides were fractionated on DEAE-cellulose or QAE-Sephadex at a flow-
`rate of 200 ml/h, according to the conditionslisted in Table I. Fractions of about 20
`ml were collected. The absorbance of every fifth fraction was measured at 250, 260
`and 280 nm. The values measured at 260 nm wereplotted versus the elution volume
`(Fig. 1). Fractions were collected within the vertical dotted lines of Fig. 1. On re-
`peated addition of pyridine,
`the volatile triethylammonium hydrogencarbonate
`(TEAB) was removed in vacuo. The pyridine was removed by co-evaporation with
`3% aqueous ammonia. Finally the remaining solution was lyophilized. The sodium
`chloride-Tris-HClI buffer was removed by gel chromatography on a Sephadex G-15
`column, In order to remove the urea, the combined peak fractions were diluted to
`1:2.5 in water and pumped on a DEAE-Sephadex column (40 cm * 2 cm) previously
`equilibrated with water. The column was washed with water until free of chloride,
`and wastheneluted with a 1 M sodium chloride solution. The oligonucleotides eluted
`with the salt were desalted by gel chromatography and lyophilized.
`High-performance liquid chromatography (HPLC) of the deprotected oligonu-
`cleotides. HPLC was performed according to the conditions summarized in Table II
`on an analytical column (250 mm x 4.6 mmm I.D.) and a preparative column (250
`mm * 8mm J.D.) equipped with a precolumn (30 mm x 8 mm I.D.) packed with
`Nucleosil 7 C,g. Experiments 1—3 of Table II were carried out at room temperature,
`4-6 at 50°C. One A260 unit of the oligonucleotide was dissolved in !—-10 ul water and
`applied to the column. The combined fractions were desalted as follows: the TBA
`solution obtained was added to 50 ml dichloromethane. A saturated aqueous picric
`acid solution was added dropwise to the stirred mixture until the aqueous layer had
`becomeslightly yellow. After separation of the layers, the aqueous phase wastreated
`with Dowex 50W-X8 (H*) and chromatographed on a Sephadex G-15 column (40
`em * 4cm). The fractions containing product were combined, evaporated to dryness
`in vacuo and lyophilized.
`HPLC of d(T4G4T4G4) after total hydrolysis by formic acid. One A269 unit of
`d(T4G4T4Ga4) was treated with 500 pl of 90%formic acid at 170°C during 45 min.
`The reaction mixture was lyophilized and dissolved in about 200 pl of 50 mM
`aqueous ammonium acetate (pH 6.8). About 0.10 Ag¢o units of this solution were
`
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`PURIFICATION OF OLIGODEOXYRIBONUCLEOTIDES
`
`167
`
`TABLE |
`
`CHROMATOGRAPHIC PURIFICATION OF PROBES (DISSOLVED IN WATER) OBTAINED AFTER THE
`DEPROTECTION OF THE PROTECTED DODECAMERS AND HEXADECAMERS USING DEAE-CEL-
`LULOSE (EXPERIMENTS 1, 2, 3a, 4) OR QAE-SEPHADEX (EXPERIMENT 3)
`The columns (diameter 2 cm) were eluted with increasing salt concentration using triethylammonium hydrogencar-
`bonate (pH 7.8) (A) or sodium chloride-0.05 M Tris-HCl (pH 7.6) + 7 M urea (B).
`
`Experi-—Deprotected Applied Column—Elution conditions
`
`
`ment
`oligonu-
`probe
`lengih
`o
`No.
`cleotide
`{Ax6o0
`fem}
`Temper-
`units/ml)
`ature
`CC)
`
`Volume (1) and
`salt concentration (M)*
`oe
`Mixing
`Reser-
`vessel
`voir
`
`
`Eluent
`
`Step
`No.
`
`**
`
`TaGaTq
`
`10 200/300
`
`50
`
`2
`
`3
`
`GaTaGa
`
`4500/150
`
`TyGaT4Gu
`
`14 500/350
`
`25
`
`25
`
`3a
`
`T4GuTyG,***
`
`3900/100
`
`25
`
`4
`
`TyGaTsGy
`
`6700/200
`
`25
`
`50
`
`25
`
`25
`
`50
`
`50
`
`I
`2
`3
`4
`
`1
`2
`3
`
`I
`2
`3
`4
`
`I
`2
`3
`4
`
`l
`2
`3
`4
`
`1.0, 0.05
`B
`2.0, 0.05
`B
`2.0, O15
`B
`1 M NaCl 1.0
`
`A
`A
`A
`
`1.0, 0.10
`2.0, 0.10
`0.5, 1.0
`
`1.0, 0.05
`B
`2.0, 0.05
`B
`1.0, 0.5
`B
`1 M NaCl 0.7
`
`1.0, 0.05
`B
`2.0, 0.05
`B
`1.5, 0.20
`B
`1 M NaCl 1.0
`
`1.0, 0.05
`B
`2.0, 0.05
`B
`1.8, 0.20
`B
`1 M NaC! 1.0
`
`_
`2.0, 0.15
`2.0, 0.30
`_
`
`2.0, 0.40
`—
`
`=
`2.0, 0.50
`
`_
`2.0, 0.20
`1.8, 0.35
`
`—
`2.0, 0.20
`1.5, 0.35
`
`* WhenBis used as the eluent M refers only to the sodium chloride concentration.
`** The total amounts of deprotected oligonucleotides were chromatographed in three experiments,
`*** Rechromatography of the mixture of d(T,4G,) and d(T,G4T4G,)isolated from experiment 3.
`
`fractionated on a Nucleosil 7 C,g column (250 mm x 4.6 mm I.D.) with 50 mA¢
`ammonium acetate (pH 6.8) as the eluent (see Fig. 5).
`
`RESULTS AND DISCUSSION
`
`The DNAfragments d(G4T4G4), d(T4G4T4) and d(T4G4T4,G,4) were obtained
`from the corresponding fully protected oligonucleotides after cleavage of the pro-
`tecting groups and chromatographic separation of the oligonucleotides. The crude
`product d(T4G4T,4), obtained after deprotection of 1.05 g dodecamer, wasfraction-
`ated on DEAE-cellulose in three portions, employing a linear increasing gradient of
`sodium chloride containing 7 M urea at 50°C, as indicated in Table I (experiment1),
`
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`

`H. SCHOTT. R. SEMMLER, H. ECKSTEIN
`
`
`aso
`
`aso
`
`030
`
`=£
`
`az
`O10
`3
`aos
`
`
`
`
`
`168
`
`oa=
`
`2
`a
`
`”
`g
`<=
`
`
`
`O40
`
`—
`030 7
`oS
`020 £“
`
`8
`aio
`oo 2
`
`040
`
`_
`030 7
`°o
`a20 £‘
`aw 6
`oo 2
`
`Elution vol,
`
`in l
`Fig. 1. Chromatographic purification of d(T,G4T4Gyq) resulting after deprotection of the protected hexa-
`decanucleotides, which were synthesized using different strategies. (a) Fractionation (experiment 3, Tables
`1, III) ofthefirst hexadecanucleotide d(T,G,T4G,4) on a QAE-Sephadex columnat 25°C with an increasing
`sodium chloride gradient, buffered to pH 7.6 by 0.05 Mf Tris-HCL (b) Rechromatographyof the mixture
`corresponding to peak II resulting from (a) on a DEAE-cellulose column (experiment 3a, Tables 1, ID)
`at 50°C with an increasing sodium chloride gradient in 7 M urea, buffered to pH 7.6 by 0.05 M Tris-HCl.
`(c) Fractionation (experiment 4, Tables.I, IIT) of the second hexadecanucleotide d(T4G4T,G,) using the
`same conditionsas in (b). Column: 25cm * 2 cm. Flow-rate: 200 ml/h. Within the dotted lines, fractions
`of peaks I and II were pooled, desalted and lyophilized.
`
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`PURIFICATION OF OLIGODEOXY RIBONUCLEOTIDES
`
`169
`
`TABLE Il
`
`CONDITIONS AND RESULTS OF ION-PAIR REVERSED-PHASE HPLC OF OLIGONUCLEOTIDES ON
`A NUCLEOSIL 7 C,, COLUMN (LENGTH 250 mm)
`
`Eluents: A = 7.5 mM tetrabutylammonium hydrogensulphate (TBA) pH 7.0: B = 7.5 mf TBA pH 7.0 in 75%
`aqueous acetonitrile, C = 5 mAf TBA pH 6.8; D = $ mM TBA pH 6.8 in 70%aq. acetonitrile.
`
`
`
`
`Experi-|Chromatographed oligonucleotides Column Elution Retention Fig.
`
`~
`-
`-
`ment
`~
`diameter
`conditions
`hme
`Yield
`No.
`Designation
`Amount
`(mum)
`fin}
`fAr60
`(%}
`units)
`
`
`
`
`d(G4T4)
`d(T4GsT4)
`d(T.G4T4)
`
`60.0
`2.5
`50.0
`
`91
`98
`94
`
`8.0
`4.6
`8.0
`
`70%A, 30%B
`50% A, 50% B
`50%A, 50% B
`
`9.23
`12.92
`12.22
`
`2a
`=
`2b
`
`d(T4Gq)
`d(GgTsGq)
`
`0.5
`0.3
`
`92
`60
`
`4.6
`4.6
`
`26,22
`31.78
`
`3a
`3b
`
`60% C, 40% D
`changed within
`48 min to
`
`d(TsG4T4Gq) 3c 0.3 9] 4.6 20% C, 80% D 36.23
`
`
`
`
`
`
`
`1
`2
`3
`
`4
`5
`
`6
`
`
`
`
`
`Of the Azo units applied to the column, 39% were due to d(T4G4T4) and 18% to
`d(G4T4) . The remaining 43%consisted of removed protecting groups and ofseveral
`oligonucleotides of shorter chain length. Fractions which contained d(T4G,4T4) or
`d(G,T,) from three experiments were pooled and worked up, thus giving 351 mg of
`dadecamers and 150 mg of octamer. On the basis of the fully protected dodecamer,
`the yield of d(T4G4T4) was 53%.
`The crude productof the second dodecamer d(G4T4G4) was also fractionated
`by means of DEAE-cellulose. When a small quantity of dodecamer d(G4T4G,4) was
`chromatographed on a DEAE-cellulose column, no clear peaks could be detected in
`the region of the dodecamer. This result was quite a surprise, especially since the
`formation of a fully protected condensation product had been confirmed by thin-
`layer chromatography. After the deprotection of 600 mg of the corresponding pro-
`tected d(G4T4G,4), preparative chromatography (experiment 2, Table I) could be
`performed only with a considerable loss of oligonucleotides. Contrary to the pre-
`viously described fractionation of d(T4G4T4), the column was eluted with an increas-
`ing concentration oftriethylammonium hydrogencarbonate buffer (TEAB) at 25°C.
`The oligonucleotide leaving the column at a salt concentration of 0.35—-0.39 M was
`identified as d(T,4G4). The required dodecamer d(G4T4G,4) wasfinally eluted by 1 M
`TEAB (see Table III). 15%of the applied Azo units were due to d(T4G4) and 20%
`to d(G4T4G,4). On working up the pooled fractions, 20 mg d(T4G,4) and 30 mg
`d(G4T4G4) were obtained correspondingto a yield of only 8% in relation to the fully
`protected dodecamer.
`This rather low yield might be explained by assuming that part of the dode-
`camer was degradated during the cleavage of the protecting groups. This explains the
`elution of numerous short-chain oligonucleotides. Another reason for the low yield
`lies in the fact that substantial losses of guanylate-rich dodecamers occur during
`chromatography. Chromatography at elevated temperature (45°C) did not increase
`
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`170
`
`TABLE Hl
`
`H. SCHOTT, R. SEMMLER, H. ECKSTEIN
`
`RESULTS OF THE CHROMATOGRAPHIC PURIFICATION (SEE TABLE I) OF THE DEPROTECTED
`OLIGONUCLEOTIDES
`
`
`lsolated oligodeoxynucleotide
`Oligonucicotide eluted
`Experi-
`
`menttecee oe woe —eenot eee -
`
`
`
`
`No.* Weight—Yield™**Sait Amount Peak Designation
`
`
`
`
`
`
`concentration oteee(Fig. 1} (mg) (%)
`
`(M)
`{A260
`(%)**
`units}
`
`
`18808
`39808
`670
`920
`1350
`4680
`
`I
`
`2
`
`3
`
`3a
`
`4
`
`0.13-0.17
`0.21-0.25
`0.35—0.39
`1.00
`0.23--0.30
`0.42-0.50
`
`0.15-0.18
`0.27-0.30
`0.14-0.16
`0.30-0.33
`
`18.4
`39.0
`14.9
`20.4
`93
`32.2
`
`Not shown
`Not shown
`Not shown
`Not shown
`I{a)
`Ha)
`
`d(G,T,)
`d(TGyT)
`d(TsGq4)
` d(GyT4G,4)
`d(T4G4)
`d(T4Gq4)
`+
`d(T4G4T4Ga)
`d(T4G)
`I(b)
`41.5
`1620
`d(T4G4T4Gq)
`II(b)
`21.0
`820
`2520
`37.6
`l(c)
`d(T,Ga)
`
` 1800 26.9 Il(c) d(T4G4T4G4)
`
`
`
`
`508
`1178
`20
`30
`40
`
`170
`
`50
`30
`70
`55
`
`52.78
`
`7.9
`
`47
`
`22.1
`
`* See Table I.
`** Based on the total amount of the probe applied.
`*** Based on the protected oligonucleotides.
`® Average of three experiments,
`
`the recovery of guanylate-rich oligonucleotides. In this case, extended elution with
`strong buffer solution resulted in a broad second peak of nucleotide material. During
`rechromatography a part of this material was eluted with the normalretention time.
`Similar problems have been reported® when purifying guanylate-rich oligonucleotides
`on Partisil 10-SAX. Even when chromatographing small amounts, other authors?
`have reported unusually low recoveries (40%) from a PEI column in the case of
`oligonucleotides containing three or more consecutive deoxyguanosine monomer
`units.
`
`the deprotected hexadecamer
`Chromatography of small quantities of
`d(T,G4T,G,) on a DEAE-cellulose column yielded no clear peaks in the region
`where octamers and longer-chain oligonucleotides are eluted. The chromatographic
`purification of larger amounts of a hexadecamer was performed as follows. A 700-
`mg amountofthe first hexadecamer, which was synthesized using only one nucleo-
`base protecting group, was deprotected and the d(T,G4T4G,4) obtained was frac-
`tionated on QAE-Sephadex with an increasing gradient of sodium chloride at 25°C,
`according to the conditions given in Tables I, III (experiment 3). The elution profile,
`shown in Fig. la, exhibited two main peaks: 9.3% of the applied Asgo units were
`contained in peak I and amounted to 40 mg d(T4G,)after isolation, peak I] contained
`32%ofthe applied A269 units and 170 mg of a mixture of d(T4G4) and d(T4G4T4G,4)
`(see Table IIT). Furthermore, the elution profile indicates two different shorter-chain
`oligonucleotides eluted previous to the octamer. These oligomeric units might be the
`result of chain degradation, occurring during cleavage ofthe protecting groups. For
`
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`PURIFICATION OF OLIGODEOXYRIBONUCLEOTIDES
`
`71
`
`the isolation of d(T4G,4T4,Gaq), the mixture corresponding to peak II was rechro-
`matographed on a DEAE-cellulose column, see Table III (experiment 3a), with an
`increasing sodium chloride gradient, containing 7 M urea,at 50°C. The elution profile
`(Fig. 1b) again showed two main peaks, with numerous smaller side peaks forming
`a high baseline. It is remarkable that oligonucleotides were still eluted from the
`DEAE-cellulose column at 50°C with | M sodium chloride, although the same mix-
`ture was eluted from the more basic anion exchanger QAE-Sephadex during the
`separation with 0.42-0.50 M sodium chloride at 25°C (see Fig. la). From the fractions
`corresponding to peak I, containing 42% of the applied A260 units, 50 mg d(T4Ga)
`were isolated. The work-up of peak II, containing 21% of the A360 units applied,
`resulted in 30 mg d(T4G4T4G4), which is only 4.7% of the fully protected hexade-
`camer.
`
`The greatest portion of the deprotected guanylate-rich oligonucleotides was
`lost during the preparative fractionation on the ion exchangers QAE-Sephadex and
`DEAE-cellulose. As clearly demonstrated in Fig. 1a, quite a large part of d(T4G4)
`is associated with d(T4G4T4G,). Therefore both oligonucleotides are eluted together
`within peak II, although the octamerdiffers significantly from the hexadecamerin
`its negative charge. By rechromatography (experiment3a, Fig.
`|b), using 7 M urea
`and a temperature of 50°C, however, d(T4G4) and d(Ts4G4T4G.4) were separated.
`Both 7 M urea and the increased temperature during the elution counteracted the
`formation of aggregates. The high baseline in the elution profile (Fig. 1b) also indi-
`cated that the mixture corresponding to peak IT in Fig. la, besides both main prod-
`ucts, contained additional oligonucleotides of various chain lengths, which are as-
`sociated with the main products. On the other hand, it cannot be excluded that octa-
`and hexadecanucleotides were also eluted in the background. Despite the drastic
`elution conditions, part of the applied mixtures was retarded to such an extent that
`it was not eluted without the use of | M sodium chloride. According to our experi-
`ence, mixtures of corresponding guanylate-poor oligonucleotides do not exhibit such
`difficulties. For example, d(G4T4) could be separated from d(T4G4T4) (see exper-
`iment 1 in Tables I, II1), although these oligonucleotides differ less in their negative
`charges in comparison to d(T4G,4) and d(T4G4T4Ga,
`The purification of a second hexadecamer, which was synthesized using an-
`other strategy, resulted in comparable results. After deprotecting 350 mg of fully
`protected hexadecamer, the solution containing d(T4G4T4G,4) was directly fraction-
`ated on a DEAE-cellulose column at 50°C with an increasing sodium chloride gra-
`dient, containing 7 M urea without any previous separation (see experiment 4, Tables
`I, III). The elution profile (Fig. 1c) generally corresponds to that in Fig. 1b, except
`that the bulk of the shorter-chain oligonucleotides was eluted prior to peak I,
`d(T,G4). Although the first hexadecamer, in contrast to the second one, was syn-
`thesized using guanylate monomer units with doubly protected guanine residues,
`bothsolutions exhibited similar percentages of short-chain oligonucleotides, after the
`protecting group had been cleaved. Because most of these side products had been
`removed during the preseparation of the first hexadecamer (experiment 3, TablesI,
`IID), they are lacking in the elution profile (experiment 3a, Fig. 1b) upon rechroma-
`tography. The fractions corresponding to peak I (Fig. 1c), which contained 37.6%
`of the applied A269 units, amounted to 70 mg d(T4G4). The work-up of peak IT,
`corresponding to 27% of the Aygo units, resulted in 55 mg d(T,G4T4G,). On the
`
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`

`172
`
`H. SCHOTT, R. SEMMLER, H. ECKSTEIN
`
`basis of the fully protected component, d(T4G4T4G4) was obtained in 22% yield,
`whereasthe yield of the hexadecamersynthesized according to the other strategy was
`only 4.1%. It cannot be excluded that the low yields result chiefly from the chro-
`matography and not from insufficient protection of the bases during the synthesis.
`The non-specific and irreversible adsorption, which caused the heavy losses of
`the deprotected oligonucleotides on the ion exchangers, occurred also on Nucleosil
`Cig which is commonly used for reversed-phase HPLC ofoligonucleotides. In re-
`versed-phase HPLC on Nucleosil 7 Cig recoveries > 90% could be achieved only
`when analytical amounts (< 3 A269 units) were applied. On applying 30 4369 units,
`only 50% were eluted within the expected region. The other part appeared in sub-
`sequent peaks or even at the end of the gradient. Especially when fractionating
`d(G4T4G,4), identical compounds had different retention times. Finally, we found
`that the totally deprotected oligonucleotides could be separated satisfactorily by ion-
`pair reversed-phase HPLC!°'!3, as described below.
`In order to remove any contamination, HPLC was performed at room tem-
`perature with a preparative Nucleosil 7 C;g column (250 mm x 8 mm I.D.) permit-
`ting up to 60 Az6o units to be fractionated. Elution of the columns was achieved by
`a two-component system (see Table I). The elution was monitored at 260 nm and
`resulted in the elution profiles shown in Figs. 2 and 3. The oligonucleotides d(G,T4)
`and d(T4G4T4) were eluted under isocratic conditions (experiments 1—3) using 7.5
`mM tetrabutylammonium hydrogensulphate (TBA) pH 7.0 as eluent A and 7.5 mA¢
`TBA pH 7.0 in 75% aqueousacetonitrile as eluent B. The oligonucleotides d(T4G,).,
`d(G4TsGy) and d(T,G4T4G,4) were fractionated with a linear increasing gradient
`(experiments 4-6), the concentration of eluent D (5.0 mM TBA pH 6.8 in 70%
`aqueousacetonitrile) increasing from 40 to 80% within 48 min. Eluent C was 5.0
`mM TBApH 6.8. The integration ofthe elution profiles (Figs. 2 and 3) showed that
`the oligonucleotides, except d(G4T,G,4), were contaminated to an extent of less than
`10%, demonstrating that
`the previous column chromatographic separations on
`DEAE-cellulose or QAE-Sephadex led to oligonucleotides of sufficient purity. The
`fractions corresponding to the main peaks within the vertical dotted lines were
`pooled, desalted and lyophilized. As a test of purity, the oligonucleotides were re-
`chromatographed in amounts of 10-15 yg at 50°C on an analytical Nucleosil 7 Cy,
`column (250 mm x 4.6 mm I.D.), e.g.. experiment 2 (Table IT) demonstrates that
`the purity of the isolated oligonucleotides exceed 98%.
`The guanylate-rich d(G4T4G,4), however, could not be purified to an extent
`beyond 60%, by ion exchange or by ion-pair reversed-phase HPLC, as could be
`concluded from the integration of the elution profile (Fig. 3b). It is possible that the
`dodecamer was of much higher purity judging from the elution profile. In support
`ofthis is the elution of identical guanylate-rich oligonucleotidesat different retention
`times, Furthermore, the dodecamercould be used successfully for enzymaticligation,
`as will be described elsewhere’. Therefore contaminations up to the presumed
`amount can certainly be excluded.
`Both the purity and sequence of d(G4T4), d(T,Gq4) and d(T4G4T4) were con-
`firmed by sequencing the oligonucleotides, carried out according to the well known
`two-dimensionalfingerprint method!*~18. Contrary to our expectation,thefingerprint
`method could not be used for the sequencing of d(G4T4G,4) and d(T,4G4T4G,). Be-
`cause of the strong adsorption of the guanylate-rich oligonucleotides on the poly-
`
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`173
`
`PURIFICATION OF OLIGODEOXY RIBONUCLEOTIDES
`
`
`
`Time in min
`
`Fig. 2. lon-pair reversed-phase HPLC of d(Gq4T4) and d(T4G414) on a Nucleosil 7 Cyg column (250 mm
`x 8mm [.D.) at room temperature underisocratic conditions (see Table IT} with a flow-rate of 2 ml/min.
`(a) d(G4T4) eluted with a mixture of 70% A and 30% B; d(T4G4T4) chromatographed with 50% A and
`50% B.A = 7.5 mAf TBA, pH 7.0: B = 7.5 mAf TBA, pH 7.0 in 75%aqueous acetonitrile.
`
`saccharide matrix, a significant separation of the partial hydrolysates of these oli-
`gonucleotides by means of two-dimensional chromatography failed, thus a finger-
`print could not be obtained. Therefore, the partial hydrolysates of the radioactively
`labelled dodecamer d([°*P]G4T4G4) and hexadecamer d((3?P]T4G4T4G4) were sep-
`arated only one-dimensionally on a polyacrylamide gel under denaturing condi-
`tions'® by meansofelectrophoresis. The separation of the twelve or sixteen spots
`confirmed that the oligonucleotides synthesized indeed correspond to dodecamers
`and hexadecamers, respectively.
`d(T,G4T4G4) was also sequenced according to the method of Maxam and
`Gilbert?° (see Fig. 4). This method is based on a specific chemical modification of
`Cyt, Cyt + Thy, Ade + Gua and Guain four parallel reactions. During the partial
`
`ic)
`
`Aaso '
`
`11i
`
`j
`40
`
`Time in min
`
`Fig. 3. lon-pair reversed-phase HPLCof(a) d(T4G,), (b) d(G4T4Ga,) and (c) d(T4G4T,G4) on a Nucleosil
`7 Cyg column (250 mm x 4.6 mmI,D.) at 50°C with a gradient of 40 to 80% D over 0 to 48 min (see
`Table I); flow-rate:
`| ml/min. C = 5 mM TBA, pH 6.8; D = 5 mM TBAin 70% aqueous acetonitrile.
`
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`174
`
`H. SCHOTT, R. SEMMLER, H. ECKSTEIN
`
`hydrolysis only the modified nucleobases are supposed to be eliminated and the po-
`lynucleotide chain should be cleaved at the point where these nucleobases are missing.
`Thepartial hydrolysate obtained is separated into fragmentsofdifferent chain lengths
`by gel electrophoresis, resulting in the autoradiogram of Fig. 4. The sequence of the
`hexadecamerfrom the 3’- to the 3’-terminal is obtained by following the most black-
`ened bands in the four lanes from the top (hexadecamer) to the bottom (monomer
`unit). The interpretation of the autoradiogram is given in the right part of Fig. 4.
`The degradation pattern confirms the sequence of d(T4G4T4Gy4). Possible failure
`sequences of synthetic oligonucleotides cannot be detected conclusively and can
`therefore not be excluded. For example, the “C lane”, to which the “Cyt degrada-
`tion” was added for control purposes, contains strong bandsin its upper part, which
`might be correlated with C contaminations. However, this is to be excluded in this
`case, because only T- and G-monomer units have been employed in the synthesis.
`The presence of other nucleobases was independently excluded by totally de-
`grading the hexadecamer chemically. Using formic acid, the oligonucleotide was de-
`graded, according to well known methods?!:?, to its nucleobases. The total hydrol-
`
` C CITAIGG
`
`'
`
`ud
`wn
`WwW
`2
`5
`2
`F
`uy$°
`-@.
`|
`
`.
`1g. ¢
`
`C CAG G
`
`—_—
`—
`—
`—
`—_
`—_—
`_
`a
`
`*pl,Gut, S,
`G3
`Ga
`G
`
`*pl4 Sal,
`T3
`Ty
`T
`
`:
`—"pla Gy
`
`come
`Gg
`=,— _G
`“pla
`8
`a
`
`=
`
`=
`—
`
`the nucleotide-spectific degraded
`Fig. 4. Left part: autoradiogram after gel electrophoresis of
`d((S*P]T4G4T4G.,) using the Maxam and Gilbert method. C, C/T, A/G, G denote C-specific, C + T
`cleavage, A + G cleavage and G-specific cleavage of the oligonucleotide. The chemically degraded oli-
`gonucleotide is fractionated on a “20% polyacrylamide gel” (0.025 cm =x 20cm * 40 cm) with 50 mAf
`Tris-borate—1 m4 EDTA buffer. Electrophoresis proceeded at 2.5 kV/6 mA for 2 h. Right part: inter-
`pretation of the sequence patterns. *p denotes [**P].
`
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`PURIFICATION OF OLIGODEOXYRIBONUCLEOTIDES
`
`178
`
`ysate was fractionated on a Nucleosil 7 C,g column by means of reversed-phase
`HPLCunderisocratic conditions. Only two peaks were obtained (see Fig. 5b). As is
`seen from the elution profile (Fig. 5a) obtained by chromatography ofthe four nu-
`cleobases underidentical conditions, the retention times (9.76 and 11.69 min) match
`those of Gua (9.77 min) and Thy (11.67 min), respectively. Having found only the
`two nucleobases expected in the total hydrolysate, the presence of other nucleobases
`within the hexadecamer synthesized can be excluded. From the integration of the
`peak areas of Fig. 5b and in view of the molar absorption coefficients at 260 nm
`(Thy, 9600 1 mol~* cm~'; Gua, 13 700 1 mol~! cm~!), a molar ratio of Thy: Gua
`of 1.02:1 was calculated. The nucleobase composition of d(T4G4T4G,) determined
`was very close to that expected (1.00:1).
`
` ¢
` Ade29.16
`
`102030 40
`Time in min
`
`Fig, 5. Reversed-phase HPLC on a Nucleosil 7 C,g column (250mm x 4.6mm I.D.)at room temperature.
`Eluent: 50 m4 ammonium acetate, pH 6.8; flow-rate, | ml/min. Applied probe: (a) the test mixture of the
`four nucleabases Cyt, Gua, Thyand Ade; (b) about 0.1 469 units ofthe totally hydrolysed d(T4G4T4Gu).
`
`CONCLUSIONS
`
`The preparative chromatography of guanylate-rich oligonucleotides, employ-
`ing different separation materials (DEAE-cellulose, QAE-Sephadex, Partisil 10-SAX
`and Nucleosil C,), can be performed only with considerable loss of oligonucleotides.
`Therefore, in the oligonucleotide synthesis there is only a limited possibility of sep-
`arating impurities using chromatography. Guanylate-rich oligonucleotides of dif-
`ferent chain lengths associate with each other, thus causing identical compoundsto
`be contained within different peaks and be eluted from the columnat different times.
`At the same time, part of the product remains irreversibly adsorbed on the ion-
`exchanger matrix. The formation of aggregates between both the oligonucleotides
`and/or their derivatives and between the oligonucleotides and the polymer matrix is
`the reason whythedesired oligonucleotide cannot be obtained when small quantities
`of condensation product are worked up by column chromatography.
`Remarkably, the dodecamer d(G4T4G,4), which couldbe purified only partially
`and characterized not unequivocally, however, resulted in the 36mer and other po-
`
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`176
`
`H. SCHOTT, R. SEMMLER, H. ECKSTEIN
`
`lynucleotides upon enzymatic ligation with the dodecamer d(T4G4T4)’. This result
`demonstrates that the oligonucleotides can be used in enzymatic reactions directly
`after their synthesis, without tedious final purification. Also that the common en-
`zymatic reactions employing oligonucleotides do not require an high standard of
`purity, because the enzymes are able to select the “fitting compound” from the mul-
`titude offered.
`The increasing demand for oligonucleotides necessitates their preparation in
`large amounts. Therefore, preparative synthesis on the largest scale possible is an
`urgent objective. In our opinion there is no need for considerable improvements in
`the strategy of the oligonucleotide synthesis, but there is a great demand for more
`efficient separation methods for purifying the oligonucleotides after the deprotection
`without major losses.
`
`ACKNOWLEDGEMENT
`
`This work was suported by the Deutsche Forschungsgemeinschaft.
`
`REFERENCES
`
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`
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