Investigation of porphyrins and metalloporphyrins by fast atom bombardment mass spectrometry

281

Analytica Chrmrca Acta, 241 (1990) 281-287 Elsevler Science Publishers B V , Amsterdam

Investigation of porphyrins and metalloporphyrins atom bombardment mass spectrometry Stephen

Naylor

by fast

* and John H Lamb

MRC Toxicology Umi, Carshalton, Surrey SM5 4EF (Great Brrtarn)

Chnstopher Department

A Hunter, of Chemrstty,

James A Cowan

and Jeremy

K M Sanders

*

Unrverslty of Cambrrdge, Cambrrdge CB2 IEW (Great Brrtarn) (Received

19th Apnll990)

Abstract The use of fast atom bombardment mass spectrometry (FAB-MS) to study a senes of porphynns (H,P) and metalloporphyrms (MetP) was mvestlgated Methods of sample preparation and choice of a smtable matnx for optimum FAB-MS studies of porphynns are described Rates of demetallatlon and the redox chenustry of such compounds were exanuned and the solution chenustry of metalloporphynns was compared with their behavlour m FAB-MS Such conslderatlons lead to an understanchng of various aspects of the physuzo-chenucal events occurnng m the FAB matnx By using FAB matrices with different electromc or ac&c properties, it was demonstrated that demetallatlon can occur either by proton &placement or one-electron reduction of the metal It was also found that demetallatlon by protons m FAB-MS correlates with the stablhty index (S,) One-electron reductions of Ag(II)P and Cu(II)P were shown to occur via an excited-state mtermechate Consideration of fragmentation and metal ion uptake by two multi-component porphynns m FAB-MS also confirmed the premously asslgned geometry of these compounds Keyword

Porphynns,

Metalloporphynns

In this paper are described methods of sample preparation and the choice of a suitable matrix for optimum FAB-MS studies of metalloporphyrms, and also demetallatlon reactions and the redox chenustry of metalloporphyrms The conclusions from these model porphyrms were used to mvestlgate the preferred conformations of two multi-component porphyrms, H,4 and H,5 Note that throughout this article structures are dlfferentlated as either the free base, e g H,2, or the metallated form, e g ZnH,4

Naturally occurring porphyrins play an lmportant role m numerous blologlcal, blochenucal and geochermcal processes [l], and mass spectral analysis of low-molecular-weight monomeric porphyrin derivatives has been readily achteved using electron-impact (EI) loruzatlon [2] and chemical lomzatlon (CI) [3] mass spectrometry However, more complex porphyrins such as cofaclal dlmers are not particularly amenable to analysis using such techniques because of their higher molecular weight and lability The “soft” lomzatlon technique of fast atom bombardment mass spectrometry (FAB-MS) has been used m analyses for a variety of polar, thermally labile compounds [4] However, FAB-MS has been utilized to only a limited extent for the study of porphyrins and metalloporphyrms [5-81 0003-2670/90/$03

50

0 1990 - Elsevler Science Pubhshers

EXPERIMENTAL

Materials

and samples

All solvents graphic grade BV

used were of liquid chromatoThe FAB-MS matrices glycerol,

282 S NAYLOR ET AL

OMU

40

1

FAB-MS

283

OF PORPHYRINS

ammoglycerol (AG), thlodlglycol (TDG), thloglycerol, tetraglyme and 3-mtrobenzyl alcohol (NBA) were purchased from Aldnch and were vacuum-dlstllled prior to use Zmc acetate was purchased from Aldnch and used without further punflcatlon The syntheses of all the porphyrms used have been described previously [9] Mass spectra

All positive-ion FAB mass spectra were recorded on one of two instruments A Kratos MS-50 mass spectrometer operating at a full accelerating voltage of 8 keV with a mass range of 10000 daltons was equipped with a standard Kratos FAB source and an Ion Tech gun Xenon was used as the pnmary atom beam, accelerated to 8 keV with an ion current equivalent to 1 PA Spectra were obtained with a magnet scan rate varying between 10 and 100 s decade-’ and the data were output to a UV stnp-chart recorder The source pressure was typically ca 1 3 X lop3 Pa (lop5 Torr) A VG70-SEQ mass spectrometer of EBQQ geometry operating at a full accelerating voltage of 8 keV with a mass range of 3000 daltons was equipped with a standard VG FAB source and an Ion Tech Gun Xenon was used as the primary atom beam, accelerated to 8 keV with an ion current equlvalent to 1 PA Spectra were obtained at a scan rate of 5 s decade-’ and the data were collected and processed using a VG 11/250 system Porphyrms were first dissolved m dlchloromethane (which had been passed through a basic alumma column to remove any traces of acid) m order to improve their solublhty m the FAB matnx Typically, ca 5 nmol of porphynn m 2 ~1 of dlchloromethane were added to 2 ~1 of matnx on the FAB probe The sample and matrix were thoroughly nuxed and subjected to FAB-MS

RESULTS

AND

slpate the energy of the bombarding fast atoms (ions) before desorptlon of the analyte mto the gas phase The crucial role played by the matnx m obtammg a successful FAB mass spectrum 1s often still underestimated The matnx must possess several properties the analyte must be soluble m the matrix, but for optimum results the analyte must have a surface excess m order to obtain abundant molecular ions [lo], the matnx must be chermcally inert towards the analyte, e g , If the analyte 1s acid sensitive an acidic matnx such a thloglycerol should not be used, and the matnx should be of low volatlhty m order to allow detection of molecular ions over a reasonable penod of time In this study a variety of commonly used matrices were used but it was found that the most abundant molecular ions were observed when either NBA or TDG was used as the matnx It should be noted that no acid co-solvent such as tnfluoroacetlc acid was added, as protons m the FAB matrix demetallate metalloporphynns (see below) Sample preparation of the porphyrm/metalloporphyrm was also found to be crucial m order to obtain an abundant molecular ion m FAB-MS The porphynn was usually introduced as a dlchloromethane or chloroform solution to aid m dlssolving the sample m the matnx Attempts to dissolve solid porphyrm directly m the matnx led to poor quality FAB mass spectra with a low signal ion abundance The importance of the solub&y of the porphyrin m the matnx was emphasized by the observation that when dlchloromethane solutions of metalloporphyrms were added to thlodlglycol with no nuxmg, and the dlchloromethane was removed by gentle heating, the metalloporphyrms precipitated and no molecular ions could be detected when the sample was subJetted to FAB-MS

DISCUSSION

Demetallatron Choice of matrix condltrons

Typically m FAB-MS, the sample 1s dissolved m a chemically inert, mvolatlle liquid matnx and then bombarded with either h&-energy atoms (8-10 keV) or ions (15-30 keV) The matnx serves both to dissolve the analyte sample and to dls-

The posltlve-ion FAB mass spectra of metalloporphynns are surprlsmgly complicated In general, the spectrum of a metalloporphyrm MetP (where Met refers to the metal and P 1s structure l), displays a quasi-molecular ion [MetPH]+, a molecular ion [MetP]+ and ions correspondmg to

284

S NAYLOR

freebase porphyrm ([H,PH]+ and [H,P]+) The dommant Ion vanes dramatically urlth the metal, the matnx and the nature of the substltuents and pendant groups on the porphyrm Protonatlon of basic sites on the porphyrm leads to quan-molecular ions (MH+, where M = MetP) The formation of radical cation molecular ions (M+‘) 1s also well documented [8] However, urlth metalloporphyrms a vanety of other reactions are also possible Protons generated m the colhslon cascade (caused by lmpactmg fast atoms) can displace the central metal ion m the porphynn macrocycle [ll] with net substitution or demetallatlon MetP + 2H++

H,P + Met*+

(1)

This results m the observation of ion species corresponding to free-base porphyrms m FAB-MS Previous work has demonstrated that an electron-nch medium 1s created m the matm Such electrons can effect metal reductions of the type Cu*++

e--+

Cu+

(2)

as described by De Pauw et al [12] Clearly, uptake of an electron by a metal ion can only occur when the ion has an accessible lower oxldatlon state For metalloporphyrms, feasible reactions include Ag(I1) + Ag(I), Cu(I1) -+ Cu(1) and Co(II1) + Co(I1) + Co(I) In a lower oxldatlon state, the metal-porphyrm interaction 1s generally less [ll] and so demetallatlon by protons (Eqn 1) 1s facilitated by a one-electron reduction Demetallatlon by protons m the matrix The stability of metalloporphyrms m solution with respect to acid demetallatlon 1s gven by the stability mdex (S, ) The validity of this theoretical quantity has been confirmed by expenment the rate or extent of acid demetallatlon m solution generally correlates with S, [ll] The stablhty of a series of metalloporphyrms with respect to acid demetallatlon m the FAB matrix was investigated and the results correlated remarkably well with S, (Table 1) In order to suppress the effects of one-electron reductions, NBA was used as the matnx, and to avoid the effects of excited-state electron-transfer reactions only the MHf signals were considered (although the trends for the M+’ signals are m fact smular) Hence demetallatlon should occur only via simple acid proton displacement (Eqn 1) Ta-

TABLE

ET AL

1

Correlatlon porphyrms

between the extent of demetallatlon and stablhty mdex (S,)

m metallo-

Metal m MetP (1)

Demetallatlon a m NBA matnx (%)

Stablhty index (S, )

WW)

92 40 23 17 4 3 1 NDb

3 64 446 4 60 5 78 6 12 8 92 715 9 25

Zn(I1) Ag(II) Co(I1) Cu(II) Ag(III) Mn(III) Co(II1) a Calculated

as ion ratio

{ [H2PH]+/([MetPH]++[H,PH]+)}

x100

Determmed for the average ion abundance of the respective moletles detected after l-2 mm m the fast atom beam b ND = no loss of metal detected

ble 1 gives the ratio of [H,PH]+ to [MetPH]+ ion abundances and the results clearly show agreement between the stability of the metalloporphynn m solution and its behavlour m FABMS Proof that It 1s protons m the matrix which effect these demetallatlon reactions was obtained from exammmg the spectra acquired using dlfferent matrices (Table 2) When AG 1s used, the concentration of available protons 1s dramatically reduced, and therefore demetallatlon should be inhibited However, one-electron reduction processes are also important m this matrix and so one must consider only metalloporphyrms m which the metal 1s not readily reduced Table 2 shows that for Zn(II)P, Mg(II)P and Co(II)P no demetallatlon was observed when ammoglycerol was used as the matrix TABLE

2

Extent of demetallatlon for a senes of monomenc (1) as a function of the FAB matrix Matnx

NBA TDG AG

porphynns

Demetallatlon d (W) of monomenc porphyrms (l), where metal M 1s Co(I1)

Zn(II)

Mg(II)

Ag(II)

Cu(II)

17 24 NDb

40 51 NDb

92 95 NDb

23 88 NA’

4 30 23

ab AsmTablel

’ NA = data not obtained

FAB-MS

285

OF PORPHYRINS

Demetallatlon by electron-transfer processes The slgmflcance of electron-transfer processes can be estabhshed by comparmg the behavlour of metalloporphyrms in TDG and NBA matrices For example, the poatlve-ion FAB-MS of Ag(II)P m TDG or tetraglyme as matnx etiblts an abundant free-base porphynn ion ([H,PH]+) at m/z = 595 {[Ag(II)PH]+ [H,PH]+= 1 12) However, addition of an electron acceptor, such as benzoqumone, to the matnx reduces demetallatlon {[Ag(II)PH]+ [H,PH]+ = 3 l} The use of the electron-accepting matrix NBA leads to a dramatic reversal of this signal intensity ratio {[Ag(II)PH]+ [H,PH]+ = 24 l} and, even after 5 mm m the xenon beam, the [Ag(II)PH]+ [H,PH]+ ratio 1s still 12 1 The mtroductlon of an electron smk m the form of the matrix has slowed the rate of demetallatlon and imphe\ that electron reduction can be important m the demetallatlon process Met(II)P

+ e-+

Met(II)P-

--) [ Met(I)P]

- 5

+ H,P + Met+

(3)

The metal m the + 1 oxldatlon state 1s complexed only weakly by the porphynn and so acid proton displacement occurs very rapidly The results m Table 2 indicate that such reduction processes are important for Ag(II)P and Cu(II)P, where a dramatic increase m demetallatlon occurs when TDG 1s used as the matnx Further evidence for one-electron reduction 1s obtamed when Zn(I1) ions are added to the FAB-MS matrix In solution, Zn(I1) does not displace Ag(I1) from a porphyrin [ll] However, m the presence of zinc acetate, the FAB mass spectrum of Ag(II)P m a TDG matrix shows abundant signals due to [Zn(II)P] + and [Zn(II)PH]+ We ratlonahze ths result on the basis of the followmg pathway

let state 2-2 2 eV above the ground state [13] This state 1s low enough m energy to be excited as it lies wthm the O-3 eV energy range, suggested by Wllhams and Naylor [14] to be populated m the colhslon cascade caused by lmpactmg fast atoms When excited, porphynns become exceedingly good electron donors and readily take part m electron-transfer reactions Such processes produce porphynn radical cations and should lead to an enhancement of the intensity of the M+ signal, le, MetP + MetP* + EA + MetP+ + [EA]-

TABLE

Ag(II)P

+ e-+

Ag(II)P -+ Zn(II)P

+ [Ag(I)P]

- zn2’i

+ Agf

(4)

In contrast to Ag(II), Ag(1) porphyrms are h&11y unstable [ll] and so the metal 1s readily displaced by Zn(I1) once reduction has occurred

3

Ratio of radical catlon to protonated ions detected for a series of Met(II)P (1) m postwe-Ion FAB-MS usmg either NBA or TDG as matrix Porphyrm

NBA

excited

smg-

TDG

[MetP]+/ [MetPH]+

H,P Exerted states Porphyrins have an electronically

(5)

where EA 1s an electron acceptor If Eqn 5 1s an important process, it should be associated with an intense radical cation signal The electron-donating capablhtles of different porphyrms (their oxldatlon potentials) should, therefore, be related to the intensity of the radical cation signal The ratios M+/MH+ for both the metallated (MetP) and demetallated (H,P) signals m the FAB mass spectra of a senes of metalloporphyrms are listed m Table 3 The stnkmg feature of these results 1s that, for metalloporphyrms, the proportion of radical cation 1s slgmflcantly larger m NBA then TDG The electron-accepting matrix clearly stimulates reactions of the type illustrated m Eqn 5 and so increases the intensity of the radical cation signal This suggests that electronically excited states are mdeed formed m the colhslon cascade In the case of Zn(II)P and Mg(II)P, these systems are not complicated by one-electron reduction processes (see above) In NBA and TDG, the ratio of radical cation to quasi-molecular ion for the free-base signal ([H,P]+/[H,PH]+) 1s essentially constant

Zi(II) Mg(I1)

[H,P]+/ [H,PH]+

[MetP]+/ [MetPH]+

02 02

07 09

03 12 15

[H,P]+/ [H,PH]+

01 01 02

S NAYLOR

286

for H,P, Zn(II)P and Mg(II)P Ths 1s further evidence that one-electron reductions do not occur for Zn(II)P and Mg(II)P However, the proportion of radical catlon mcreases dramatically for the metallated signals compared with the free-base signals The oxldatlon potential of Zn(II)P and Mg(II)P 1s ca 0 8 eV compared with 1 0 eV for H,P, 1 e , the metallated denvatlves are better electron donors Hence the excited-state electrontransfer reactions shown m Eqn 5 occur more rapidly and the observed proportion of radical cation Increases on metallatlon Tlus again suggests that electronically excited states are generated m the colhslon cascade Mass

spectral

analysis

of

multr-component

porphyrins

The two multi-component porphyrins H,4 and H,5 were also analysed by FAB-MS, and the results are summarized m Table 4 The fragmentation of H,5 1s conceptually slrmlar to that of BPC2 (correspondmg to H,2) The posltlve-ion FAB-MS gave a molecular ion, MH+, at m/z = 1509 plus fragment ions m the range m/z 10801000 and 500-400 (Table 4) The m/z 1080-1000 fragments correspond to porphyrin dlmers, 1 e , simple loss of pyromelhtmude moiety The m/z 500-400 fragments are monomeric porphyrin units correspondmg to loss of the pyromelhtmude and a

TABLE

4

Ratlo of molecular eon abundance for both H,4 and H,5 and blsmetallated, monometallated molecular Ions plus ratlo of molecular Ion abundance and fragment Ion regions m/r 11501000 and 550-400 a +

Por-

MH+

phyrm

[Zyl]+

[ZnHl+

H,4 ZnHP H,5 ZnH,S

_

_

ND‘ 121

3 1 _ 11

MH+ h -fragment Ions m/z 1150-1000

MH+ ’ fragment Ions m/z 550-400

12 1 115 1 12 1 7 1

ND‘ 9 1 8 1 8 1

a All ratios determmed m posltwe-ton FAB-MS usmg TDG as matrix after the samples had been m the fast atom beam for 1 mm ’ Fragment Ion abundance determmed by measurmg the most abundant Ion cluster m the mass range under conslderanon ‘ ND = not detected above background even after 5 mm m fast atom beam

ET AL

porphyrm moiety The negative-Ion FAB-MS showed only fragment Ions m the m/z 400-300 correspondmg to the pyromelhtmude region, group Hence electron transfer reactions lead to the formation of a porphyrm radical cation (or a porphyrm dlmer radical cation) and a pyromelhtmnde radical amon Addition of a small amount of zmc acetate to the matnx (either NBA or TDG) containing H,5 resulted m uptake of the metal cation m accord with our results for the simple cofaclal dlmers (H,3) (Addition of zmc acetate to H,3 m NBA matnx afforded ions m posltlve-ion FAB-MS corresponding to the metallated species MetH,3 and Met,3 After 1 mm m the fast atom beam the H,3 MetH,3 Met,3 ratio was 0 2 1 2 2) Ions correspondmg to the uptake of both one and two metals were observed almost immediately on addition of the metal ions to the matIuc The fragment ions observed for the metallated species, MetH,S and Met,S, were smular to those from the parent free-base compounds Fragment ions were observed m the regions m/z 1200-1060 (dlmers) and m/z 560-440 (monomers) The posltlve-ion FAB-MS of H,4 was remarkably different from that of H,5 (Table 4) An abundant molecular ion MH+ was observed at m/z = 1453, but no low-mass fragment ions m the range m/z 500-400 were detected The major fragment ions were detected m the range m/z 1080-1000, attributable to porphyrin dlmers, corresponding to simple loss of the pyromelhtmude group Again, the negative-ion FAB-MS showed only fragment ions correspondmg to the pyromelhtumde moiety m the region m/z 360-240 Addltlon of zmc acetate to the matrix containing H,4 agam resulted m uptake of the metal cation However, a molecular ion correspondmg to uptake of only one metal was observed The blsmetallated species was not detected, even after 10 mm m the fast atom beam In addition, the fragment ions observed for the metallated derivative, MetH,4, were surprlsmgly different from those from the parent free-base compound (Table 4) MetH,4 exlublted fragment ions m the region m/z 560440, correspondmg to a zmc porphyrin monomer There were no fragment ions due to monomeric free-base porphyrins wluch would be observed ca

FAB-MS

287

OF PORPHYRINS

60 daltons lower The abundance of fragment Ions m the range m/z 1080-1000, due to the porphyrm dlmers which dommated the H,4 fragmentation pattern, was slgmflcantly reduced (Table 4) The behavlour of these two complex porphyrin adducts 1s ratlonahzed on the basis of then stereochermstry and photophyslcal properties, and an m-depth analysis will be reported elsewhere Nevertheless, previous spectroscopic analysis suggests that H,4 and H,5 adopt substantially different conformations m solution [15], and these metallatlon studies provide strong evidence that the conformatlonal preferences depicted m the structures are indeed correct H,4 can only be metallated once, showing that the central porphyrin m the “sandwich” 1s stencally shielded Expenmental and theoretical studies of ~7-7 mteractlons predict that the two bulky aromatic groups (porphyrm and pyromelhtmnde) will n-stack with the central porphyrm [16] The geometry of these mteractlons 1s such that the aromatic moieties prefer to he over the cavity at the centre of the porphyrm r-system, so blockmg the second metal bmdmg site In any case, the bndgmg chams are so short that ths molecule has very hnnted flexlblhty Clearly, the geometry of H,5 1s radically different it adopts an “open” conformation so that metallatlon of both porphynns 1s easy The authors thank SERC (JAC, JKMS) financial support

the MRC (S N , J H L), and DEN1 (CAH) for

REFERENCES

Freeman, 1 A R Fersht, Enzyme Structure and Mechamsm, Readmg, 1985, J Dlesenhofer and H Mlchel, Angew Chem , Int Ed Engl , 28, (1989) 829, G Eghngton, m A L Burlmgame and N Castagnoh, Jr (Eds ). Mass Spectrometry m the Health and Life Sciences, 1985, p 41 R Sot London, Ser A, 293 2 AH Jackson, Phdos Tram (1979) 21 and A A 3 G J Van Berkel, G L Ghsh, S A McLuckey Tumman, J Am Chem Sot , 111 (1989) 6027 4 C Fenselau and R J Cotter, Chem Rev, 87 (1987) 501 D Kessel and C K Chang, Blamed 5 B D Musselman, Envuon Mass Spectrom , 15 (1988) 257 6 J Mel11 and J Selbl, Org Mass Spectrom , 19 (1984) 581 T J Wllhams and J E Campana, Blochem I L Kurlanslk, Blophys Res Commun , 111 (1989) 478 and D H Wd8 R G Brereton, M B Bazzaz, S Santlkarn hams, Tetrahedron Lett , 24 (1983) 5775 Sot, Perkm 9 J A Cowan and J K M Sanders, J Chem Tram 1, (1987) 2395, (1985) 2435 10 S Naylor, A F Fmdels, B W Gibson and D H Wdhams, J Am Chem Sot, 108 (1986) 6359 11 J W Buchler, m K M Srmth (Ed ), Porphynns and Metalloporphynns, Elsevler, Amsterdam, 1972 12 E De Pauw, G Pelzer, J Manen and P Natahs, m A Bennmghoven (Ed ), Ion Formation from Orgamc Solids, Spnnger, Heidelberg, 1986, pp 103-107 B Pearce, G S Beddard, J A Cowan and 13 R J Harnson, J K M Sanders, Chem Phys , 116 (1987) 429 14 D H Wdhams and S Naylor, J Chem Sot, Chem Commun , (1987) 1408 15 J A Cowan, J K M Sanders, G S Beddard and R J Harnson, J Chem Sot , Chem Commun , (1987) 55 16 CA Hunter, J K M Sanders and A J Stone, Chem Phys , 133 (1989) 395