Application of a weakly basic dimethylamino-modified silica ion exchanger to the separation of oligonucleotides

CHROM.

12,184

APPLICATION

OF

A

WEAKLY

SILICA ION EXCHANGER

BASIC

DlIMETHY LAMINO-MODIFIED

TO THE SEPARATION

OF OLP~NUCLEOTIDES

W. IGST and K. K. UNGER Irrstiti fZ

Anorganircire m-d Aruzlytkhe Chemie, Jdmmzes Gutenkerg-UtaiversitZt, 6500 Moinr

(G.F.R.)

and R LIPECKY htsth~

fw

and & G. GASSEN

Organixke

Ckemie, Tecknticke E?acksckde, 6100 Danmtadt

(G.F.R.)

SUMMARY

LiCbrosorb RR-S, RR-18 and Dial as well as a newly synthesized basic dimethylamino-m&ed silica ion-exchanger @MA-silica) were applied for the separation of adenylic acid, cytidylic acid and uridylic acid oiigoribonucleotides. On LiChrosorb m-8 and RR-B, respectively, in aqueous buffered eluents (K$R?O.,E&PO,), the retention of oligonucleotides was increased with decreasing number of nuckotide units in the solute, i.e., with increasing hydrophobic character_ The elution behaviour of oligonucleotides on LiChrosorb Diol followed the same order but took place according to a size-exclusion mechanism. The retention of oligonucleotides on DMA-silica is assumed to be based on electrostatic interactions between ffie charged solutes and the ionic surface sites of DMA. This is evidenced by (i) the exponential decrease of k’ of the solute with increasing salt concentration of buffer, and (ii) the increase of log k’ of the solute with the number of nucleotide units, i.e., negative charges in the oligoribonucleotide solute. The relative contribution of ionic and moiecular interactions to total retention of the solutes is discussed.

Considerable interest has centred on the separation and analysis of mono-, ohgo- and polynucleotides since these are the components of deoxyribonucleic acids (DNAs) and ribonucleic acids (RNAs)’ respectively. The separation of mononucleotides by high-performance liquid chromatography on reversed phase as well as on ionexchange packings was reported in a series of paper&“. Oiigonucleotides are usually chromatographed on alkylammonium ion-exchangers bass OQpolysaccharides, but this is very time-consuming”- 18_The analysis time could be drastically reduced on columns packed with pellicular weak anion exchangers and a concentration gradient lQs20, e-g-, uridyhc acid oligonucleotides up to the heptadeca compound were eluted within 70 mm=. In this work a totally porous microparticulate silica ion exchanger with bonded

groups is applied to the separationof oligoadenylicacid (oligo(A)), oligccytidylic acid (oligo(C)) and oligouridylicacids (oligo(U)) ribonucleotides.In order to examine the retentionmechanismof oligonucleotidesin more detail_two other chemicallybonded microparticulatesilicas, LiChrosorb RP-18 and Dial, were employedas packingss. dimethyhmino

PackGzgs

LiChrosorbSi 100 (particlediameter5 pm; E. Merck, Darmstadt, G.F.R.) was modifiedwith(1 ,%eopxy-3-propylpropoxy)trimethoxysilane purchasedfrom Dynamit Nobel, Troisdorf,G.F.RZ’. The productwas treatedwith reagentgradeN,N-dime& ylethanolamine(E. Merck). The tizl packing designatedas DMA-silica is believed to exhibit the surfacestructure: H

=Si-(CH&O-CH,-C-CH,OH I

I

OH

0

The surfaceconcentrationof the functionalgroupsis 2.0 ~mol/mz.The theoreticaJ exchange capacity derived from the nitrogencontent was calculatedto be 0.78 mecmiv./g. LiChrosorb Diol, R&S and RP-18 (all Spm) were commercialproducts from E. Merck Chromarographic studies The chromatographwas a Hewlett-PackardModel 1010 A fitted with a W

photometerof 254 nm wavelength.Columns were300 x 4 mm I.D. The eluentwas a phosphatebufferconsistingof K,HPO, and HsPO, adjustedto pH = 7.0. The phosphate concentration,HPG,2-, was varied from 0.01 to 0.27 &f. The dimer up to the pentamerof oligo(U) werepurchasedfrom &&ringer, Ingelheim,G-F-R. All other oligoribonucleotidesused in this study weresynthesizedby Lipeckyand Gassen. Abbreviations are used to designate the oligonucleotidest2.The bases are abbreviatedas adenine(A), uracil (U) and cytosine (C). The correspondingnucleosides are adenosine,uridineand cytidine,respectively.A nucleotideis writtenas Np whereNp means the nucleotideunit comprisingthe base, the pentose and the phosphate group, e.g., (Up),U means a pentanucleotidemade up of four Up units and a terminatinguridiue. RESULTS

AND

DI8CUSSION

Retention of Oligoribonudeotides on reverse&p/me packing Since the oligonucleotidesare strong acids, the proton of the phosphatebridge

is dissociatedat pH 7.0. It is expectedthat the compoundswill be retardedweaklyon reversed-phasepackings in an acetonitrile-watereluent owing to their pronounced

hydrophilic character. Indeed, on LiChrosorb RI-18 and with acetonitrikwater (8Q:ZO to 20:80) the uridine was found to be retained slightly wherezrsthe ohgorihonucleotides(oligo(U)) were eluteclbefore uridineas the referencesubstance.No appreciableeffect on retention was observed when the water in acetonitrif~water (20330) was replacedby a solution of K,HP04 and H3P04 (pH 7.0) and the salt concentrationwas variedfrom 0:05 to 0.25 M. In purelyaqueous buff& solutions(0.02 IW K,HPO,-H,PO,, pH 7.0) the capacity factor of oligo(C) decreasedwith increasing number of nucleotideunits in the range betweenCpC and (Cp)&. This elution behaviour can be explainedby the fact that the polar characterof the solute increases with the numberof nucleotideunits. Under theseconditionsthe capacityfactor r-ange was O-2. Longer retentionmight be achievedby increasingthe salt concentrationbut such studieswere not done. In conclusion, oligoribonucleotidescan be separatedon reversed-phasepackingsin the elution order of increasinghydrophobiccharacter. Retention of oligoribomcledides on dial packfng The LiCbrosoFb Diol carriesbonded groups of type:

*

=Si-
OH

I

I

OH OH

As eiuents, phosphate buffer solutions of pH 7.0 were employed containingvarious HP0,2- concentrations.Again, the chromatographicstudieswereperformedwith the ohgo seriesas solutes. At a given HPO,‘- concentrationthe solutes are elutedin the sequence: (Up),v < (Up),U < (Up).U < UpU c midine Increasingthe salt concentrationfrom 0.01 to 0.23 M increasesretentionfor a given ohgo( but all solutes are still eluted before to, indicatinga type of size-exclusion~ mechanism(see. Fig. 1). Becauseof the limited separationrange of the packing only (Up)&, UpU and uridine could be resolved completely. Retention of oligoribonucleotides on BMA-silica

When conditioned with buffer solution the DMA-silica 8s a basic anion exchanger. Since the p& value of the base cation is around 9-11 the terminating dimethylamino group is protonated at pH G 7.0. A simple calculation shows that a high populationof silanol groups stillexistsat the modifiedsurface,as welias orgauofunctional groups. The surface concentmtion, Q,-,=. of hydroxyl groups on native silica before reaction is CQ.8.0 ymol/mz. The resultingsurfaceconcentrationof the organic moiety is calcultedto be ca. 2.0 pmoI/m*. Assuming a bifunctionalreaction of the modifier, 2 - 2.0 = 4.0 I.cmol/m2of hydroxyl groups undergoesreaction and 8.0 - 4.0 = 4.0 pmol/m2 does not react. In addition,one methoxygroupper modiier molecule remainsunreactedand is converted,after bonding through hydrolysis,into a silanol group. Its concentration’also equals2.0 ~mol~mz,so that total concentration of 6 pmoI/m* of hydroxyl groups’is expectedto be presentat the modifiedsurface. In aqueous solution the silanol groups act as a weak acidic cation exchanger,

406

W. JGST,

IL

K. UNGER,

R

LIPEL=,

EL 43. GASSEN

i

2.0

! U

LIPU UJPUJ

WPW

I

WPl$

Fig.l_ Retention volume of uridine and oligotidyiic acids on a LiChrosorb Diol coIumn as fun+ tion of *he n-xmber of nuckotide units at various HpoIz- concentrations of the phosphate buffer_ Conditions: column, 300 x 4 mm I.D.; packing, LiChrosorb Dial, 5 ,um; eluent, K2H?%-HQO, (PH 7.0). 0. 0.01 M; q , OSk5M; 77. 0.11 M; A, 0.23 M; detector, W at 254nrn_

r&easing a proton and forming a negatively charged siloxane group. Several models were proposed to explain the deprotonation properties of silanol groups at the silica surfacp3. By applying different methods such as infrared spectroscopy, titration, etc. the pll, of silanol groups was estimated to be 7.1 * 0.5’“*6. This value relates to unmodified silica and the question arises as to what extent the pK, is infiuenced by bonding organ0 functional groups at the silica surface. Considering the newly formed hydroxyl groups bonded at the silicon atoms introduced by the modifier, the adjacent propyl group behaving as a weak electron donor is abIe to induce a charge to the silicon atom. This charge accumulation reducesthe polarization of the dipole of the hydroxyl group and hence may decrease the pK, of his type of hydroxyl group. On the other hand, the unreacted hydroxyl groups, like those at native silica, should show a more pronaunced acidity due to (d-p)z conjugation effects with neighbouring siloxane bonds?. For comparison, the pK, of monosilicic acid is estimated to be ca. 9.9 (ref_ 28). Since the DMA-siiica columns is run at pH 7.0 it is believed that no more than half of the hydroxyl groups (6 pmol/m’) are deprotonated. It is of interest to consider the effect of these negative surface chafges on the retention behaviour in addition to that of the positive dimethylammonium groups. As already discusseci by Knox and PrydG9 for short-chain aminopropyl silica, a neutralization of both groups may occur, provided that they are mobile enough to be attached closely. This effect seems to be highly improbable in our case since the organic chain should possess a high degree of rotational mobility. Assuming a linear extended organic chain in DMA-silica, the distance between the positively charged terminating dimethylammonium group and the negatively charged SiO- group fuzd to the silica Surfacemay be calculated to be CQ. 1.5 nm, which is fairly hkrge.&cause

IEC

OF OLIGONUGLEOT’EDES

407

ofthefavourable steric position of the dimethylammoniumgroup, it is considered to be most active in interactions with charged solutes, particularly when they are as large as the oligoribonucleotides. Discussing an ion-exchangemechanismfor retentionof ribonucleotideacids on DMA-silica, we have again a special situation becausethe nucleotidesare polyvalent ions or polyelectrolytes.In addition, as for RNA, *Jleoligoribonucleotides exhibita primarystructureand a type of secondarystructurel_The primarystructure is representedby the nucleotidebackbone comprisingthe phosphatebridge and the pentose with the attachedbase. The secondarystructureis determinedby the spatial arrangementof the nucleotidechain, which may form either an orderedhelix with base stackingor a disorderedrandom coil. The conformationof oligoribonucleotides has beenexaminedin severalpapers3o-32.Oligo(U) are reportedto have a low tendency to stack and henceshow no orderedstructureat room temperature3°s31, whileoligo(A) form maiuly heliceP. In the helix structurethe sugarphosphatebackbonesare pointed outwardswith one negativecharge per phosphate,while the stackedbases form a hydrophobic coreU. According to the number of nucleotide units, the oligoribonucleotideacids show a high negativecharge which is compensatedby surrounded positive comer ions. Thus, it appears that electrostaticinteraction with the ionic functional groups is possible through multisite attachment. The strengthof interaction seems to be dependentnot only on the nominal total charge of the oligoribonucleotidebut also on the secondarystructureof the acid. The fundamentalaspectsof the ion exchangeof polyelectrolytessuch as type of interactions,thermodynamicequilibria,effectof temperature,etc. are reviewedby FeitelsoP and Marinsky35.Based on the work of Bernardi-o on the elution behaviour of DNA and RNA fragments, oligonucleotidesand nucleoside polyphosphates on hydroxyapathecolumus, Kawasaki’1-53 made extensive calculations on the chromatographicbehaviour of macromoleculeswith rigid structureson hydroxyapatitecolumus. In the firsttwo papers%42 he developedthe theoryfor the chromatography of macromoleculeson hydroxyapatiteboth under static and dynamic couditions. In the following publications63-uhe discussesspecial cases. In order to determinethe type of retentionmechanism,the concentrationof phosphateeluent,C, was variedand the capacityfactors(k’) of solutesweremeasured. In the case of an ion-exchangemechanismthe capacityfactor of a solute falls exponentially with increasingCS. The correspondingplots for oligo(U) and oligo(Aj, respectively,given in Figs. 2 and 3, show the expectedbehaviour. It should be emphasized that the k' valuesof the respectivenucleosides(uridine and adenine)are scarcelyintIuencedby the salt concentration.By plotting the logarithmof k’ vs. the number of nucleotideunits for oligo(U) and oligo(A), respectively,at constant salt concentration,straighthues are obtained (Figs. 4 and 5). As expected,the logarithm of k’ increaseslinearlywith the number of negativechargesin oligoribouucleotides. As shown in Fig. 6, this relationship also holds for long-chain oligoribonucleotides, e.g., up to the heptadecaoligomes of o&o(A)_ The resultsobtained reveal that an ion-exchangeis predominanton DMA-silica. BernardiS’, in his study of nucleic acid retentionon hydroxyapatitecolumns, also suggestedan ion-exchangemechanismbetweenthe phosphate groups of polynucleotidesand the calcium ions of the packing with no direct interventionof the base and the sugar residue.In other papees -O he concludedthe following elution

W. JOSr,

‘403

K. K, UNGER,

RLEPECECY, H- G. GASSEN

k' I

I

I

k'

I

f IO

I.0

Ql

-

(Ml

cHmzc

2 mdence of k’ ofoEgo(U) on the HEW.?- concentrationof the eluent_Conditions: cohumn, 300 x d mm IS.; packin& DMA-silica (5 pm); eluent, phosphatebuffer (pH 7.0); sow-mte, l-0 ti min; detector, UV at 2S4 MI. Solutes: V, uridine; q, UpU; 0, (UphU; A. (UpMJ; 0. CJpkU-

Fig.

Fig_3. Dependence of k’ of oligo(A) on the HE%*Fig. 2. Solutes: 0, adenosine; m, Ap.4; U, (A&A;

concentration of the duent. Conditions as in A, (AhA; V. tAphA.

k’

k’

t

t 10

1

9.1

03

r

I t

Fig_ 4. Dependence of K of oIigo(U) on the munber of nuckotide units. Conditions as in Fig. 2. \J, 0.23 M; m, 0.17 M; 0,0.115 M; A, 0.06 M; 0, 0.OL M. Eluent cm=-: Fig_ 5. Dependence of K of o&o(A) on the number-of nucleotide units_ Conditions as in FG 2 M; 0, 0.115M; A, 0.06 M; 0.0.01 i%f. Elutnt CT-*- : 8,0.28 M; v, 0.23 M; 1,0.17

410

W. JOSr, K. K. UNGER,

R WEc=Ky.

IL G. GASSEN

k’

kg

t 1.0

c

- LO

- LO

LO

0

-

2.0

tog l/cH&-

-

log

1rc,,,;-

Fig. 7. log k’ of oligo(U) vs. log l/c-z-_ 0, C-JPMJ; CL (UPMJ; a, WPW-

Conditions as in Fig. 2. sOIutes: m, UpU; V, (Up),U;

Fig. 8. log k’ of oIigo(A) vs. log l/C-,.*-.

Conditions as in Fig. 2 Solutes: 0. ApA;

CL (APIA

A,

0, (AP)~A;

(AP~CA.

be easily seen in Fig. 3 that the k’ value of adenosine remains generally unafI=cted by the salt concentration and aiso equals 0.1, as shown in Fig. 8. For uridine (see Fig_ 2) a slight dependence of k’ on the salt concentration is observed and k' approximates v~!ues between 0.02 and 0.04 which corresponds to that extrapolated at C = 1 M in Fig. 7. The curves in Figs. 7 and 8 can be described by

logk’ = a log lfc_:-

+ log b

where o is the slope of the function and b the ordinate is a measure of the contribution ofmolecular interaction to total retention. It appears that the slope of the straight lines includes the number of negative charges, 2, of the oligoribonucleotides as a variable. For a given nuckotide ,Z equals the number of nucleotide units per molecule minus one. For a given series such as ohgo the nominal value of a, however, is not equal to 2 bat differs by a constant factor, p. This factor p is calculated to be 0.42 & 0.2 for ohgo and 0.36 f 0.2 for ohgo( The constancy ofp witbin a given series of ohgoribonuckotides shows that k’ is directly proportional to the total nominal charge of the solute. The observed-fact that the negative charge is not equal to 2 but remarkably lower can be explained by a protection of the charge of the respective oligoribonuckotide and/or the fixed ionic sites of the D&IA-silica. The factor p, termedthe protection factor, is smaller for oSgo(A) than for ohgo( A plausible explanation for this

IEC

OF OLZGONUCLEORDES

Fig. 9.

Separation of a homologous series of oIigo(u). Conditions as in Fig. 2, exceptCm’-

0.115M and cm 4 = (Up).U;

= speed 300mm/h.timpounds: 1 = m-i&e, UpU; 2 = (VpMJ; 3 = (vp),U;

5 = (Up&U;

6 = (U&U;

7 = @J&U;

g = (VPMJ.

difference may be that the base adetie in oligo(A) is larger than uracil in oligo(u) and therefore leads to a higher protection. In conclusion, at a given phosphate buffer concentration we found less reten-

tion for o_!igo(U) than for oligo(A) on DMA-silica for the same number of nucleotide units. An analogous pattern was observed by BernardFO for poIy(U) and poly(A) on hydroxyapatite columns. Separation of ok’goribonucleotides on DMA-silica As demonstrated in Fig. 9, separation of oligo(v

ca~zbe completed within 20 min under isocrztic conditions. The method enables the biochemist, when synthesizing oligonucleotides, to monitor rapidly the composition of a reaction mixture and to derive some kinetic parameters. REFERENCES W. Guschelbauer, Nucleic Acid Structure,Springer, New York, Heidelberg, B&in, 1976. J. R M&sic and Ph. R Brown, J. Ckonrato~.., 142 (1977)641. S. C Chen and Ph. R Brown, L Chromatogr.Sci., 15 (1977) 218. I. J. ECirkknd,3. Chromatogr. Sci., 8 (1970) 72. F. S. Anderson and R C. Murphy,J. Chromatogr.. 121(1976)251. 6 Ch_ Gsret, A. L. Pogolorti and D. V. San& L&U.!.Btuckenz.,79 (1977) 603. 7 R A. Henry, J. A. S&nit and R. (3. Wm, J. Ciwomatogr.Sci., 11 (1973)358. 8 J. X EGym, J. W. Bynum and E. VoMn, AnaC.B&hem., 77 (1977) 446. 9 Nucleic Acid Constituents6y Liquid Chromatography,Varian, Walnut Creek, calif., 1970. 10 V. I. Drobishev, S. E. Mansurova and I. S. Kulaev,J. Chrom&ogr.,69 (1972)317. 11 R A. Hartwickand Ph. R Brown, J. Chromotogr..,112 (1975) 651. 12 EL R Mattkvs, Ekr. J. Bioaiem, 7 (1968) 96. 13 R Cerng, E. Cermi and J. HL Spencer, L Mol. RX, 46 (1969) 145. 1 2 3 4 5

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24 25 26 27 28 29 30 31 32 33 34 35

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H. G. GASSEN

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Wiiy-lnter-