Characterization of cisplatin adducts of oligonucleotides by fast atom bombardment mass spectrometry

ANALYTICAL

193,6-15

BIOCHEMISTRY

(1991)

Characterization of Cisplatin Adducts of Oligonucleotides by Fast Atom Bombardment Mass Spectrometry’ LeRoy

B. Martin

Department

Received

III,

of Chemistry,

August

Anton North

F. Schreiner, Carolina

and Richard

State University,

B. van Breemen’

Raleigh,

North

Carolina

27695-8204

16, 1990

The products of the reaction of the antitumor drug cisplatin (cis-diamminedichloroplatinum(I1)) with four oligonucleotide tetramers, d(GpCpGpC), d(GpGpCpC), d(TpGpApT), and d(TpGpCpT), were separated by gel permeation chromatography and characterized by negative- and positive-ion fast atom bombardment (FAB) mass spectrometry. Fragment ions indicating the oligonucleotide sequence and the position of cisplatin binding were observed in MS/MS spectra following collisional activation and B/E-linked scanning. Positive-ion FAB MS/MS spectra were characterized by platinumcontaining product ions. Nonplatinated sequence ions and internal fragment ions were present primarily in the negative-ion spectra. The most prominent fragment ions containing platinum were [HB, - Pt. B,H]+ and [HB, - Pt. B,H]+, where B, , B,, and B, were bases in the oligonucleotide tetramer, one of which was usually guanine. Both singly and doubly charged platinum complexes were observed, probably indicating reduction of Pt(I1) during the FAB ionization process. The location of the platinum complex bound to each oligonucleotide sequence could be determined, and the binding sites observed by mass spectrometry were similar to those previously determined by other methods. FAB ionization with collisional activation and MS/MS analysis could serve as a new method for structural analysis of platinated oligonucleotides. o 1991 Academic press, IIIC.

The cytotoxic antitumor drug cisplatin (cis-diamminedichloroplatinum(I1)) (1) has been shown to covar This material is based upon work supported in part by the North Carolina Biotechnology Center. Preliminary results were presented at the 37th ASMS Conference of Mass Spectrometry and Allied Topics, May 21-26, 1989, Miami Beach, Florida. ’ To whom correspondence should he addressed at Department of Chemistry, Box 8204, North Carolina State University, Raleigh, NC 27695-8204.

lently bond to and modify DNA strands both in vivo and in vitro. Several reviews of the structure and biochemical action of cisplatin have recently been published (2). In aqueous solutions, including cells, a labile chloride ligand is replaced by water, leaving a very reactive coordination site. Open coordination sites have been shown to bind to many Lewis bases, which in the cell nucleus on a DNA chain are primarily exposed nitrogens on the bases (3). The predominant site of attachment at physiological pH is the N7 of guanine, although other sites such as Nl and N7 of adenosine and the N3 of cytosine have been observed to bond to platinum. To be singly bound, the platinum complex loses one of the labile water or chloride ligands; for the platinum to be bound twice to a DNA molecule, it needs to lose both labile ligands. Both singly and doubly coordinated platinum complexes retain the two ammine groups. Platinum binding effectively impedes DNA replication and therefore cell reproduction (4), and recent evidence shows that DNA repair mechanisms are hindered (5). Although platination of cancer cell DNA is believed to the mechanism for antitumor activity, the common severe side effects of cisplatin administration have been attributed to platination of DNA in nontarget organs, tissue, and especially blood proteins (4,6). Cisplatin and other platinum(I1) complexes are currently the subject of active research directed toward reducing toxicity while maintaining high antitumor activity. The cisplatin-DNA complex that is considered responsible for most of the antitumor activity shows bidentate binding of the platinum to two adjacent guanines on the same chain (intrastrand) (7). Bonding to guanines separated by another nucleotide on the same chain (8) and complexation with other bases can also contribute to the antitumor activity. Interstrand binding between two guanines is possible and has been observed in vitro, but not at levels high enough to be pharmacologically active (7). The active complexes in each

6 All

Copyright 0 1991 rights of reproduction

0003-2697/91 $3.00 by Academic Press, Inc. in any form reserved.

MASS

SPECTROMETRY

OF

PLATINATED

case, however, consist of at least two nucleotides. Because distinct changes in DNA helix geometry such as unstacking and melting are at least partly responsible for the inhibition of synthesis and repair, an even longer length of the DNA chain is involved in the antitumor activity of the cisplatin-DNA complex. The analysis of oligonucleotide rather than mononucleotide adducts will extend the understanding of the platinum-bound active site. Platinated oligonucleotide adducts have been separated and analyzed by HPLC (9,lO) and characterized by ‘H, 31P, and lg5Pt NMR (11,12), ir spectroscopy (13), and X-ray crystallography (14). Kozelka et al. (15) have carried out extensive molecular mechanics modeling based on NMR and crystallographic data. Compared to these and other biochemical techniques, analysis of nucleotides by mass spectrometry requires less time and smaller amounts of analyte. Mass spectra are typically acquired in a few minutes, and sample requirements are often as low as nanomoles for small oligonucleotides. The low volatility of underivitized oligonucleotides precludes their analysis by electron ionization (EI)3 or chemical ionization (CI) mass spectrometry. Analysis of nonmetalated oligonucleotides by FAB mass spectrometry has been briefly summarized (16). Due to the presence of acidic phosphodiester linkages with pK,‘s less than 1, nucleotides are strong acids and are typically observed as deprotonated molecules, [M - HI-, in negative-ion fast atom bombardment (FAB) mass spectra (17). Although both negative and positive ions of intact, nonmetalated dinucleotides have been observed (18), negative-ion FAB ionization with collisionally activated dissociation (CAD) has been used most often for sequencing and structural analysis of oligonucleotides (19,20). Compared to a FAB matrix consisting of pure glycerol, addition of a strong base to glycerol or use of neat triethanolamine enhances the formation of [M - HI- ions during negative-ion FAB mass spectrometry (21). Positive-ion FAB mass spectra have been obtained by addition of para-toluenesulfonic acid to the glycerol matrix in order to prevent loss of the acidic protons from the phosphate groups and to add an additional proton so that [M + H]+ ions could be observed (18). Because trimethylsilylation increases the volatility of nucleotides, these derivatives show lower detection limits by FAB mass spectrometry than underivatized oligonucleotides (22). However, trimethylsilylation complicates identification of fragment ions and hence structure and sequence determination (23). FAB mass spectra of a few complexes of platinum with only bases and platinum with only mononucleo-

3 Abbreviations used: EI, electron tion; FAB, fast atom bombardment; sociation.

ionization; CI, chemical ionizaCAD, collisionally activated dis-

OLIGONUCLEOTIDES

7

sides have been reported, i.e., platinated guanine and deoxyguanosine (24) and platinated bis(nucleobase) compounds (25), but not of platinated oligonucleotides. Also, EI and CI mass spectra of synthetic platinumbound nucleoside compounds have been reported (26). In this study we present the FAB mass spectrometric characterization of four platinum-bound DNA tetramers, d(GpCpGpC), d(TpGpApT), d(TpGpCpT), and d(GpGpCpC).

MATERIALS

AND

METHODS

The oligonucleotides d(GpCpGpC), d(TpGpApT), d(TpGpCpT), and d(GpGpCpC) were synthesized using an Applied Biosystems (Foster City, CA) Model 380A automated DNA synthesizer with ,&cyanoethylphosphoramidite triester chemistry. Phosphoramidites and controlled-pore glass supports were obtained from American Bionetics (Hayward, CA). Oligonucleotides were removed from the solid support by concentrated aqueous ammonia and lyophilized. After resuspension in water, lyophilization was repeated to remove residual ammonia. Completed oligonucleotides were purified by reversed-phase chromatography and stored as the dry ammonium salt. Triethanolamine, diethanolamine, glycerol, 3-nitrobenzylalcohol, and cis-diamminedichloroplatinum(I1) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Dimethylsulfoxide was purchased from Pierce Chemical Co. (Rockford, IL). High-purity water was prepared using a Millipore (Bedford, MA) Mini-Q water system. Platination of oligonucleotide tetramers was carried out by reacting approximately 100 pg oligonucleotide with a IO-fold molar excess of cisplatin (5 mg/ml) in deionized water. After reacting for 3 days in the dark at 37”C, the mixture was freeze-dried and stored at -5°C until further analysis or purification. Under these conditions, platinated oligonucleotides have been reported to be stable (27). Gel permeation chromatography of the platinated oligonucleotide was carried out by low-pressure chromatography using a Sephadex G-15 column (5 X 430 mm) with a mobile phase consisting of 1 mM ammonium acetate at a flow rate of 250 pl/min. Sample elution was monitored at 254 nm by an Isco (Lincoln, NE) Model UA-5 absorbance detector. Alternatively, HPLC gel permeation chromatography separation was carried out using a Synchrom (Lafayette, IN) Synchropak GPC-60 column with 1 mM triethylamine acetate and a flow rate of 300 pl/min. The HPLC system consisted of a Waters (Milford, MA) Model 501 pump, Rheodyne (Cotati, CA) 7125 injector, and a Waters Model 481 uv absorbance detector set at 260 nm. Chromatographic bands were collected, freeze-dried, and then analyzed by mass spectrometry. Columns were flushed thoroughly between

8

MARTIN,

SCHREINER,

injections in order to remove cisplatin, which could become extensively trapped in the gel. Mass spectra were obtained using a JEOL (Tokyo, Japan) HXllOHF double-focusing mass spectrometer equipped with a DA5000 data system and B/E-linked scanning. Xenon fast atoms at 7 kV were used for ionization. The accelerating potential was 10 kV, and the resolving power of the instrument was adjusted to between 1000 and 2000. Sodium iodide in glycerol was used for calibration of negative-ion FAB mass spectra, and cesium iodide or a mixture of cesium iodide and potassium iodide was used for calibration of positive-ion spectra. Samples were prepared by placing 2-3 ~1 of aqueous oligonucleotide sample containing approximately 5 pg onto a l-p1 matrix on a stainless-steel probe. For negative-ion FAB, the matrix consisted of neat triethanolamine (28). Mixtures of glycerol with triethanolamine or with diethanolamine were used as matrices for positive-ion FAB. B/E-linked scanning, a type of MS/MS measurement in which the magnetic field B and the electric field E are scanned to maintain a constant ratio B/E, was carried out using CAD to enhance fragmentation. For CAD, the pressure of the helium collision gas in the first field free region was adjusted so that the precursor ion intensity was attenuated 70%. Between 6 and 12 scans were averaged to obtain each mass spectrum. RESULTS Analysis

AND

DISCUSSION

of Oligonucleotides

Two of the unmodified oligonucleotide tetramers, d(GpGpCpC) and d(GpCpGpC), are self-complementary and therefore exist as dimeric hexa-anions in neutral aqueous solutions. The other two tetramers, d(TpGpApT) and d(TpGpCpT), are not self-complementary and exist primarily as monomeric tri-anions. However, neither positive- nor negative-ion FAB mass spectra of these compounds contained multiply charged molecular ions. Instead, abundant deprotonated molecules, [M - HI-, or protonated molecules, [M + HI+, were detected. For comparison to the mass spectra of platinated oligonucleotides, B/E-linked scans of deprotonated and protonated nonplatinated oligonucleotides were carried out using CAD. Figure la shows the B/E-linked scan following CAD of the [M - HI- ion at m/z 1164 of the unplatinated tetramer d(TpGpCpT). These product ions are similar to those observed for the other synthetic oligonucleotides used in this study and to those previously reported (20,29). Complete sequence information was obtained, with cleavage at each of the phosphodiester bonds and some of the fl-glycosidic bonds, particularly at the 3’ terminus (Table 1). Negative-ion B/E-linked scans were dominated by ions ending in negatively charged phosphates, e.g., [M - H-d(T)OH]at

AND

VAN

BREEMEN

m/z 939 and [d(pT)OH]- at m/z 321 (Fig. la and Table 1). A second series of ions was detected 18 mass units below that of the primary set of sequence ions and was occasionally of greater abundance (Fig. la and Table 1). The origin of the la-mass-unit difference may be attributed to dehydration of a phosphate, because ions lacking a terminal phosphate were not accompanied by the lower mass fragments. Similar fragmentation was reported for diribonucleotides by Cerny et al. (20) and was attributed to loss of water from the 2’-position of the ribose group. Because the deoxyribonucleotides used in our study lacked the hydroxyl group at the 2’-position, we suggest that dehydration occurred at the terminal phosphate. These and other important sequence ions are presented in Table 1. B/E-linked scans of [M + H]+ ions following CAD contained sequence information similar to that of the negative-ion spectra. Examples of fragment ions detected in these B/E-linked scans are presented in Table 2. Compared to the negative-ion spectra in Table 1, CAD of protonated oligonucleotides formed more abundant low mass fragment ions such as the bases [Cl’ at m/z 110 and [G]’ at m/z 150 (data not shown) and internal ions like [d(HpGpH) + H]+ at m/z 428 in Fig. 1B. In addition to an extra proton being present to form a positively charged molecule, two additional hydrogen atoms were transferred to these internal product ions during their formation and probably were bound to phosphate groups as phosphoester bonds were broken. In some B/ E-linked scans, ions corresponding to fragments of diethanolamine matrix clusters that were isobaric with the protonated oligonucleotide molecule were observed, i.e., mlz 213, 1060, and 1074 in Fig. lb. Isolation of Platinated Oligonucleotides Following reaction of each oligonucleotide with cisplatin, platinated oligonucleotides were separated from unreacted cisplatin by gel permeation chromatography. Open-bed and HPLC gel permeation chromatography produced chromatograms that were qualitatively similar. The chromatograms were reproducible, but the retention times lengthened slightly as the sample size was increased. In general, a sharp band eluted immediately after the solvent front, followed by a second sharp band, then a large broad band. For example, the chromatogram of platinated d(GpCpGpC) contained two sharp peaks at 8.7 and 12.1 min, and the final broad band was partially resolved into two at 31.6 and 36.7 min (Fig. 2). Because all these bands absorbed uv light at 260 nm and cisplatin has no absorbance at this wavelength, they contained either oligonucleotides or platinated oligonucleotides. After separation by gel permeation chromatography, the bands were collected, concentrated, and then analyzed by FAB mass spectrometry. The early eluting

MASS

SPECTROMETRY

OF

PLATINATED

9

OLIGONUCLEOTIDES

a

R e

321

650

[M-H]1164

939

1 [M-H-&(;)OH]-

U n

[M-H-d(T)OH-H20]921

d

WWW-W’l-

a

n C

\

368

3

\

[d(pCpT)OH-HpO]592

e

kW-J-~W-

WWpVWl-

\

408 I

200

300

400

[M-H-TH]’ 1038

I

6E

500

I‘2DEOA+H]+ 213

e

l

5-

T i v e Fi

HO-

4-

[MH-d(TpG)OH]’ 612

b u z a n

3-

C

e

2-

d(T)OH]+ 225

IdWpW&)+W+

6E

4i0 FIG. 1. B/E-linked scans - HI- and (b) the protonated

[MH-d(HpT)OH-CH]+ 732 [MH-d(pT)OH]+ 845 f

I

of d(TpGpCpT) molecule [M

following + H]+.

FAB

ionization

and CAD

showing

fragment

ions

of (a) the deprotonated

molecule

[M

10

MARTIN,

SCHREINER,

AND TABLE

Fragment

Assignment [M-H][M-H-B4H]o [M-H-d(B,)OH][M-H-d(B,)OH-H,O][M-H-B,H][M-H-d(B,)OH][dG%pB~KW[d(B,pB~)OH-WItdbB,pB,)OW-

[d(pB,pB,)OH-I-WI[dbB,PW

[d(pB,)OH-I-V-)[WW%p)l[WW%p)-H,Ol[dWW,p)l[dU-bB,pbH,Ol-

Ions

of [M

-

HI- Precursors by B/E-Linked

d(TGAT)

d(TGCT)

1188 1062 (36)*

1164 1038 (30) 939 (85) 921 (30) 1038(30) 939 (85) 650 (33) 632 (37) SlO(lO0) 592 (41) 321 (50) 303(10) 426 (31) 408 (44) 386 (1.0) 368 (33)

963 (100) 945 (33) 1062 (36) 963 (100) 650 (52) 632(38) 634 (95) 616 (52) 321 (31) 303 (29) 426(29) 408 (36) 410 (29) 392 (33)

1174 964 (100) 946 (73) 1023(77) 924 (80) 675 (71) 657 (67) 595 (86) 577(64) 306 (81) 288 (68)

.

d(GCGC)

1166 1040(24) 941(59) 1040(24) 941 (59) 652(35) 612 (100) 225 (30) xx xx

1176 1065 (61) 967 (100) 1025(84) 926 (98) 677 (65) 597 (59)

xx xx

1176 1065 (48) 967 (48) 1025(78) 926(100) 637 (66) 637 (66) 210 (75) xx xx

xx

xx

xx

B,H]+ B,H]+

[HBB.

‘=Pt

* B,H]+

xx

WNH,), d(GCGC)

1401

1401 1290 (60) 1192 (28)

610(100)

595 (100)

635(100)

321 (39)

321(37)

306 (32)

306 (56) 288 (35) 386 (53) 368 (60) 426(63) 408(65)

426 408 386 368

386 (26)

normalized

of Nucleotides and Scanning Following

d(GGCC)

. ‘*‘Pt. . “‘Pt.

1391

-

to the most

abundant

(21) (37) (31) (19)

fragment

*

ion in each

2

d(TGCT)

[HB, [HBZ

[d(BJOHl+

WNH,), d(GGCC)

analysis, which was not possible using the crude reaction mixture. Bands collected from sharp chromatographic peaks could be dried to white solids that were readily soluble in water and produced mass spectra with excellent signalto-noise ratios. Broader peaks that eluted later had a yellow color indicative of unreacted cisplatin. These samples were sparingly soluble in water and glycerol,

d(TGAT)

1190 1064 (8)* 965 (26) 1064 (8) 965 (26) 652(g) 636(100) 225(15) XXd xx

[M+H]+ [MH-B,H]+ = [MH-d(B,)OH]+ [MH-B,H]+ [MH-d(B,)OH]+ [MH-d(B,pB,)OH]+ [MH-d(B,pB,)OH]+

-

634 (100)

Platinated CAD

Pt(NH,f, * Assignment

WNH,), d(TGCT)

Obtained

1191 (50)

tetramer, d(5’-B,pB,pB,pB,-3’). in parentheses and has been

of [M + H]+ Precursors Obtained by B/E-Linked

Nucleotides

1415

1023 (33) 924(54) 635 (100) 617 (52) 635 (100) 617(52) 306 (27) 288(25) 386(22) 368 (1.6) 426(33) 408 (29)

408 (68) 386 (73) 368 (69)

Platinated CAD Pt(NH,L d(TGAT)

1174 1063 (31) 964 (71)

TABLE Ions

1

d(GCGC)

sharp bands contained predominantly mono-platinated oligonucleotide. The later eluting broader bands corresponded to bis-platinated species. Mass spectrometric analysis indicated that both mono- and bis-platinated species were present in every band, although gel permeation chromatography had greatly enriched each band with respect to one species. The purity of each fraction was sufficient to facilitate FAB ionization and MS/MS

Fragment

BREEMEN

of Nucleotides and Scanning Following

d(GGCC)

’ B, denotes the 3’ terminal base in an oligonucleotide b The relative abundance for each m/z value is shown column.

VAN

(TGAT)

1417 1291(75) 1193 (93) 1291(75) 1192 (93)

472 (39) 481(100) 240 (35)*+ 456 (35)

Nucleotides

WNH,), (TGCT)

WNH,), (GGCC)

1393 1268 (56) 1169 (100) 1268(56) 1169(100) 880 (32)

1403 1292 (100) 1194 (45) 1251(43)' 1153 (31)

225 (21) 472 (46) 457(74) 228 (18)2' 432 (42)

210 (43) 497 (25) 457 (29)

o B, denotes the 3’ terminal base in an oligonucleotide tetramer, d(5’BlpBapBapB4-3’). *The relative abundance for each m/z value is shown in parentheses and has been normalized to the most column. c Mass also corresponds to a fragment of a matrix cluster ion that was isobaric with the precursor ion. d Not applicable for nonplatinated oligonucleotides.

WNH,), . d(GCGC)

1403

864 (100) 864 (100) 457 (62) 457 (62) 457 (62) abundant

fragment

ion in each

MASS

Retention

SPECTROMETRY

OF

PLATINATED

time, minutes

FIG. 2. HPLC chromatogram showing gel permeation separation of platinated d(GpCpGpC). The mobile phase was aqueous 1 mM triethylamine acetate at a flow rate of 300 &min. The bands at 8.7 and 12.1 min correspond to monoplatinated oligonucleotides. The bands at 31.6 and 36.7 min contain primarily bis-platinated oligonucleotides.

insoluble in triethanolamine, and produced few ions during FAB. This might have been an ion suppression effect, in which the surface density of insoluble cisplatin was so high that desorption of oligonucleotide ions was prevented. Unreacted cisplatin on the FAB probe also had the undesirable effect of promoting cluster ion formation, which increased the chemical noise and hindered selection of sample precursor ions for MS/MS that were free from matrix clusters. The chromatographic separation of mono- and bisplatinated oligonucleotides was greater than that expected based only on size exclusion, unless the addition of a second platinum diammine moiety produced profound changes in the apparent size of the oligonucleotide. A more probable explanation is that separation mechanisms other than gel permeation were occurring on the column. Chromatographic resolution of the mono-platinated oligonucleotide bands and the bis-platinated oligonucleotide bands into two bands each might indicate different isomers. However, each pair of bands could not be distinguished by mass spectrometry. Analysis

of Platinated

Oligonucleotides

Although a matrix of triethanolamine or diethanolamine facilitated the formation of deprotonated nucleotides during negative-ion FAB mass spectrometry, this matrix was unsuitable for platinated nucleotides because of poor solubility. Addition of glycerol to triethanolamine increased the solubility of platinated nucleotides and therefore the abundance of [M - HI- and [M

OLIGONUCLEOTIDES

11

+ HI+ ions for these compounds in the FAB mass spectra. An equal volume mixture of triethanolamine and glycerol was superior to either pure triethanolamine or pure glycerol for all analyses. Negative- and positive-ion FAB mass spectra of all four mono-platinated oligonucleotide tetramers were obtained (Tables 1 and 2). The molecular ion region of each platinated oligonucleotide was characterized by multiple isotope peaks due largely to the isotopes of platinum (Fig. 3). This isotope pattern was repeated in fragment ions corresponding to the sequential loss of the two labile ammonia ligands coordinated to each platinum (Fig. 3). Platinum has six naturally occurring isotopes: 190 (O.Ol%), 192 (0.8%), 194 (32.9%), 195 (33.8%), 196 (25.3%), and 198 (7.2%). For MS/MS analysis of platinated oligonucleotides, the most abundant ion in the isotopic envelope of the protonated or deprotonated molecule was typically selected as the precursor (Fig. 3). Because partial reduction of platinum probably occurred during FAB, the most abundant ion in the isotopic envelope probably contained contributions from more than one platinum isotope. For example, the ion at m/z 1403 in the positive-ion FAB mass spectra of singly platinated d(GpGpCpC) and d(GpCpGpC) (Fig. 3a) was probably a mixture of the protonated molecule, [M + HI+, containing lg5Pt(0); the radical cation, M+*, containing lg6Pt(0); and the cation, M+, containing lg6Pt(I). At unit resolution these species would be indistinguishable. Furthermore, B/E scanning with a double-focusing-sector mass spectrometer (as opposed to tandem, four-sector MS/MS) has the additional limitation that precursor ions are selected at less than unit resolution. Therefore, contributions from m/z values slightly above and below that of the selected precursor were transmitted during B/E-linked scanning. Although both positive- and negative-ion MS/MS spectra of mono-platinated oligonucleotides contained fragment ions indicative of the oligonucleotide sequence and location of platination, the most abundant fragment ions in the negative-ion spectra originated from the nonplatinated nucleotide sequence following the elimination of platinum, i.e., [d(pB,)OH]and [d(pB,pB,)OH]in Table 1 and m/z 306 and 595 in Fig. 4. These fragment ions localized the primary sites of platination to the first two bases in each oligonucleotide, one of which was guanine. This observation is consistent with negative charges residing on the phosphate groups of the oligonucleotide and no charge or else positive charge(s) being located on Pt(O), Pt(I), or Pt(I1). Elimination of a platinum complex would leave a negatively charged fragment ion that could be detected in a negative-ion scan, but the neutral or positively charged platinum complex would be lost. Platinated fragment ions and some sequence ions were evident in the positive-ion B/E-linked scans of platinated oligonucleotides. For example, the most

12

MARTIN,

R e

;

SCHREINER,

AND

VAN

BREEMEN

a

[M+H]+ 1403 ‘,“,“I

80-

t

i v

70

e 70-

40

Theoretical

!

1403

1 1

I

II

II

60 60 30

b"

20

U

10 0 lulil

r; 60a n

I II T---;;r.

[M+H-P(NH.J]+ ,I

1370

1360

1380

1400

1390

1410

100

lb

R

e 1

90-

M-H] 1401

F

; 80e R 70. b u

[M-H-NH$

; 60a

1

[M-H-2(NH3)]

; 50e 40

1360 FIG. 3. Molecular isotope distribution

1365

1370

1375

1380

1385

ion regions of d(GpCpGpC) * Pt(NH,),, by (a) positive-ion at a resolution of 3000 is shown in the inset of (a).

1390

1395

or (b) negative-ion

FAB

400

1405

mass

spectrometry.

The

theoretical

MASS

SPECTROMETRY

OF

PLATINATED

13

OLIGONUCLEOTIDES [M-H]1401

R e

Y-

1 a

t

i v e

T-C

I

7-

A b u

G-

ii a

5

n

d(W)OHl306

C e

306

8-

4-

WWW-Wl406

WWWI

/ 426

400 FIG. 4, Negative at m/z 1401.

B/E-linked

scan of monoplatinated

800

600 d(GpGpCpC),

abundant fragment ions of platinated oligonucleotides were formed by loss of unplatinated terminal nucleosides such as [MH-d(B,)OH]+ or [MH-d(B,pB,)OH]+ (Table 2 and Fig. 5). In addition to sequence ions, a series of fragment ions corresponding to platinum-base complexes were observed. Previously, platinum-base complexes and platinum-mononucleoside complexes have been reported (24), but not as fragments generated from oligonucleotides. The most abundant fragment ion observed in this series consisted of a singly charged Pt complexed to the bases B, and B,, or B, and B, (Table 2). For example, [HT * ls5Pt *GH]+ at m/z 472 and [HG - ls5Pt * CH]+ at m/z 457 were detected in the positive-ion B/E-linked scan of (NH,),Pt * d(TpGpCpT) in Fig. 5. Other ions in this series are summarized in Table 2 as [HB, . ls5Pt +B,H]+ and [HB, - ls5Pt - B,H]+. In all of the oligonucleotides examined in this study, a guanine was located in either the 5’terminal or the internal position closest to the 5’ end. Therefore, platinum-base complexes containing guanine were much more abundant than similar dinucleotide fragments from the 3’ end. This was expected since platinum binds preferentially to guanine. Nevertheless, [HB, - ls5Pt - B,H]+ ions such as [HC +ls5Pt * TH]+ at m/z 432 (Fig. 5) and

obtained

1000 using

FAB

ionization

1200 and CAD

1400 M/Z of the deprotonated

molecule

[HA. ls5Pt - TH]+ at m/z 456 were detected, although at lower relative abundances than the corresponding complexes containing guanine (Table 2). In B/E scans of [M + H]+ ions, elimination of a negatively charged oligonucleotide moiety sometimes led to the formation of doubly charged platinum-nucleoside complexes. An example is the ion at m/z 335, corresponding to [Pt(NH& + dT + dG12’ in Fig. 5. In addition to having a narrow peak width, this ion could be identified as doubly charged by the sequential losses of ammonia ligands observed at m/z 326.5 and 318. In singly charged species, elimination of ammonia molecules would form fragment ions detected at 14-mass-unit intervals (i.e., Fig. 3). However, sequential loss of ammonia from doubly charged ions would form fragment ions differing by only 8.5 mass units, which cannot be explained by fragmentation of singly charged species. The singly charged platinum-containing fragment ions (i.e., [HB * Pt * HB]+ species in Table 2) that were detected in B/E-linked scans typically contained no ammonia ligands. These ammonia ligands were eliminated during CAD. Occasionally, a fragment ion contained a water ligand, which replaced chlorine on cisplatin and might also replace an ammonia ligand. Platinum forms

14

MARTIN,

SCHREINER,

AND

VAN

BREEMEN [M+H]+ 1393

R e '

N\Hz /NH,

[MH-d(T)OH]’ 1169

1169

E

t"

i V e

c .

f L

U Li a

L

[HG.‘VtCH]+ > 457

Pt+V’)sl’ 249 \

[MH-d(pT)OH]+ 1073

n C e

225

-

[HT.‘Vt.GH]+ 472

‘I:

ld(VHl+ 225 326.51

)

llllllL

200 FIG. 5. ml2

Positive

B/E-linked

400 scan of monoplatinated

8b0 d(TpGpCpT),

obtained

1000 using

FAB

ionization

12i30 and CAD

14b0 M/Z

of the protonated

molecule

at

1393.

stable, hydrated complexes, an example of which might be [Pt(H,O),]+ at m/z 249 in Fig. 5. However, ions containing platinum with water ligands (weighing 18 mass units) are difficult to distinguish from those with ammonia (weighing 17 mass units) due to the wide platinum isotope distribution discussed above. In contrast to the singly charged species, doubly charged ions almost always contained platinum complexed with ammonia, water, or both. CONCLUSIONS

Presented here are the first mass spectra of platinated oligonucleotides, formed by the reaction of cisdiamminedichloroplatinum(I1) with oligonucleotide tetramers. Analysis by FAB mass spectrometry showed that Pt binding occurred to bases in adjacent nucleotides, and one of these was usually guanine. Fast atom bombardment of platinated oligonucleotides produced both positive and negative ions, [M + H]+ and [M - HI-. Positively charged fragment ions consisting of Pt-base, Pt-nucleoside, or Pt-nucleotide complexes were found to contain one or two charges, indicating that reduction of platinum from Pt(I1) to Pt(1) and/or Pt(0) may have occurred during ionization.

Negative-ion B/E-linked scans of product ions from [M - HI- following CAD produced primarily nonplatinated sequence ions, and the positive B/E-linked scans with CAD of [M + HI+ ion precursors formed abundant platinated fragment ions. Overall, platination decreased the abundance of sequence ions (Table 1). Also, sequence ions were seldom observed past the first base B,, probably because of platinum bonds bridging adjacent bases (B,-B,, or B,-B,). The structures of these Pt-oligonucleotides assigned by mass spectrometry agree with those previously determined by other methods. We conclude that both positive- and negative-ion FAB mass spectrometry from a mixed matrix show great promise in the analysis of modified oligonucleotides. ACKNOWLEDGMENTS The authors acknowledge Mr. Keith lina Department of Genetics Molecular sis of the oligonucleotides used in this

Everett Biology study.

and the North CaroCenter for the synthe-

REFERENCES 1. Rosenberg, B., van Camp, don) 205,698-699.

L., and Krigas,

T. (1965)

Nature

(Lon-

MASS

SPECTROMETRY

OF

PLATINATED

2. (a) Urnapathy, dijk, van S. J. and

P. (1989) Coord. Chem. Rec. 95,129-181. (b) ReeJ., Fichtinger-Schepman, A. M. J., van Oosterom, A. T., and de Putte, P. (1987) Struct. Bonding 67, 53-90. (c) Lippard, (1987) Pure Appl. Chem. 59, 731-742. (d) Sherman, S. E., Lippard, S. J. (1987) Chum. Reu. 87, 1153-1181.

3. Horacek, P., and Drobnik, J. (1971) Biochim. Biophys. Acta 254, 341-347. 4. Roberts, J. J., and Thompson, A. J. (1979) Prog. Nucleic Acid Res. 5.

Mol. Biol. 22, 71-133. Lepre, C. A., Chassot, Biochemistry 29,811-823.

L., Costello,

C. E., Lippard,

S. J. (1990)

11. Fouts, C. S., Marzilli, L. G., Byrd, R. A., Summers, M. F., Zon, G., and Shinozuka, K. (1988) Znorg. Chem. 27, 366-376. L. G. (1985) Znorg. Chem. 24, 242112. Miller, S. K., and Marzilli,

2425. 13. Okamoto, K., Benham, V., Theophanides, T. (1987) Znorg. Chim. Acta 135,207-210. 14. Sherman, S. E., Gibson, D., Wang, A. H.-J., and Lippard, S. J. 15.

(1985) Science 230,412-417. Kozelka, J., Archer, S., Petsko, Biopolymers 26, 1245-1271.

16. Schram, K. H. (1988) Trends Anal. Chem. 7,28-32. 17. Grotjahn, L., Frank, R., and Blocker, H. (1982) Nucleic Acids Res. 10,4671-4678. 18. Moser, H., and Wood, G. W. (1988) Biomed. Enuiron. Mass Spectram. 15,547-551. 19. Cerny, R. L., Tomer, K. B., Gross, Anal. Biochem. 165,175-182.

Lippard,

S. J. (1987)

L. (1987)

862-864.

22. McCloskey, 23.

J. A. (1985) in Mass Spectrometry in the Health and Life Sciences (Burlingame, A. L., and Castagnoli, N., Jr., Eds.), pp. 521-545, Elsevier, Amsterdam. Weng, Q.-M., Hammargren, W. M., Slowikowski, D., Schram, K. H., Borysko, K. Z., Wotring, L. L., Townsend, L. B. (1989) Anal. Biochem. 178, 102-106.

24. Puzo,

G., Prome, J.-C., Mass Spectrom.

Biomed.

Macquet, J.-P., 9, 552-556.

and Lewis,

I. A. S. (1982)

25. Claereboudt, 26.

J., De Spiegeleer, B., Lippert, B., De Bruijn, E. A., and Claeys, M. (1989) Spectros. Znt. J. 7, 91-112. Roos, I. A. G., Thompson, A. J., and Eagles, J. (1974) Chem. Biol. Interact. 8, 421-427.

27. Johnson, Biol.

N. P., Hoeschele, Interact. 30, 151-169.

28. Grotjahn, trom.

G. A., and

M. L., and Grotjahn,

20. Cerny, R. L., Gross, M. L., and Grotjahn, L. (1986) Anal. Biochem. 156,424-435. 21. Sandstrom, A., and Chattopadyaya, J. (1987) J. Chem. Sot. Chem. Commun.,

6. Reedijk, J. (1987) Pure Appl. Chem. 59, 181-192. 7. Pinto, A. L., and Lippard, S. J. (1985) Biochim. Biophys. Acta 780,167-180. 8. Marcelis, A. T. M., den Hartog, J. H. J., and Reedijk, J. (1982) J. Amer. Chem.Soc. 104,2664-2665. 9. Riley, C. M., Sternson, L. A., Repta, A. J., and Slyter, S. A. (1983) Anal. Biochem. 130, 203-214. 10. Eastman, A. (1982) Biochemistry 2 1,6732-6736.

15

OLIGONUCLEOTIDES

29. Crow,

J. D., and Rahn,

L., Frank, R., and Blocker, Zon Phys. 46.439-442.

H. (1983)

R. 0. (1980) Znt. J. Mass

F. W., Tomer, K. B., Gross, M. L., McCloskey, Bergstrom, D. E. (1984) Anal. Biochem. 139, 243-262.

Chem. Spec-

J. A., and