Tandem mass spectrometric studies of porphyrins


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Tandem mass spectrometric studies of porphyrins structural and photochemical studies
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ix, 168 leaves : ill. ; 29 cm.
Laycock, John D., 1966-
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Porphyrins   ( lcsh )
Mass spectrometry   ( lcsh )
bibliography   ( marcgt )
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Thesis (Ph. D.)--University of Florida, 1994.
Includes bibliographical references (leaves 157-167).
Statement of Responsibility:
by John D. Laycock.
General Note:
General Note:

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University of Florida
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Full Text







In loving memory of Robert Cole Laycock.


There are many to whom I am greatly indebted for helping me to find the

inspiration, gumption, and courage to complete this dissertation. Many are not

included here; they know who they are -- I wish them the best.

I want to first thank my late grandfather, Robert C. Laycock, to whom this

thesis has been dedicated. He was always interested in understanding what I

was learning in my studies of science and chemistry. He gave me much

encouragement during my first year of graduate school. I miss our days of sailing

his boat out in Pensacola Bay.

I wish to express my deepest gratitude to my parents, Bob and Brigitte, for

constantly providing me with loving guidance, wise advice, and helping hands

throughout my studies. They have always been there when I needed them;

without their help, I doubt I could have completed this dissertation. I wish to

thank my sister, Maren, for talking me through my hardest times; in many ways

our spiritual growths have coincided. I am sure we will continue to grow together

for many years. I thank the rest of my extended family for their gracious

encouragement and support.

I want to thank my fellow (former) graduate students Drs. John Pike and

Nathan Yates for inspiring me to find the meaning behind putting forth a

consistent work effort. On the other hand, I want to thank John for knowing when

it is time to let the beer flow and Nate for knowing when it is time to go surfing.

I hope to continue our friendships for many years to come.

I would like to express my gratitude to Dr. Richard A. Yost for his support

and professional guidance (and understanding) throughout my graduate studies.

I thank Dr. J. Martin E. Quirke for our energetic conversations on porphyrin

chemistry during our many "porphyrin weekends"; I admire his boundless

enthusiasm for porphyrin research. I thank Dr. Jodie Johnson for training me in

the use of the TSQ 45 and for always being willing to help with troubleshooting

and other experimental problems.

I would like to thank the Yost Group members (old and new) for their

friendship and for their helpful advice with many of the aspects of preparing this

dissertation. Finally, I wish to acknowledge the members of my committee, Drs.

Willard Harrison, James Winefordner, William Weltner, and Anthony Randazzo.


ACKNOWLEDGEMENTS ..................................... iii

ABSTRACT .......... ................................. viii


1 INTRO DU C TIO N ......................................... 1

Tandem Mass Spectrometry .................................. 2
Porphyrins ... ............... .. .. .... .. .. ..... ... .... 6
Historical Perspective .................................. 7
Inorganic Chemistry of Porphyrins .......................... 9
Biological Chemistry of Porphyrins ......................... 12
Geological Chemistry of Porphyrins ........................ 15
Porphyrins of Biomedical Importance .......... ............ 18
Mass Spectrometry of Porphyrins ............................ 19
Electron ionization (El) .................................. 20
Chem ical Ionization (CI) .................... ........... 24
Tandem Mass Spectrometry (MS/MS) ...................... 25
Scope of Dissertation .................................... 26

MIXTURES ............................................. 29

Introduction ....................... ................... 29
Geoporphyrin Skeletal Types ............................. 30
Analysis of Geoporphyrins .............................. 33
Experim ental ......................................... 35
Samples ................. ......................... 35
Methods ................. ......... ............... 35
Metal Insertion ....................................... 36
Results and Discussion .................................. 37
Comparison with the Daughter Ion Spectra of Molecular
Ions of Geoporphyrins ................................. 47
C conclusions ........................................... 55


Introduction .............
Spectroscopic Studies of Nitroporphyrins ....
Mass Spectrometry of Nitroporphyrins ......
Experim ental ...........................
Compound Preparation .......
Synthesis of NO, porphyrins ....
Synthesis of isotopically labelled porphyrins
Synthesis of Ni(ll) and Co(ll) 5-NO, OEP .
Synthesis of Zn(ll) and Cu(ll) 5-NO2 OEP
Synthesis of Mg(ll) 5-NO2 OEP .........
Synthesis of Ag(ll) 5-NO2 OEP .........
Synthesis of Pd(ll) 5-NO, OEP .........
M ethods .....................
Results and Discussion ...................
Analysis of Free-base 5-NO2 OEP .........
Analysis of the Divalent Metal Complexes of 5-N
Spectroscopic studies ...............
Mass Spectrometric studies ...........
Conclusions .......................... ..




Introduction .......................
Photochemistry ..................
Photosensitizing Reactions ..........
Porphyrin Photosensitizers ..........
Clinical Photodynamic Therapy (PDT) ..
On-line Solution Chemistry ..........
Thermospray ionization ...............
Instrum entation ..................
Theory ........ ...........
On-line Flow Cell Development .. ......
Commercially Available Flow Cells .....
Residence Time Requirements .......

. .



High Pressure Photochemical Reaction Cell

Experimental ....................................... .. 120
O n-line Setup ...................................... 121
O ff-line Setup ...................................... 121
Compounds ........................................ 125
Methods .......................................... 128
Results and Discussion ....... ............ ... ........... 129
Initial TSP/MS of Porphyrins ............................. 129
On-line ................................ .......... 130
Off-line .............. ..... ........................ 144
Conclusions ........... ............................. 151

5 CONCLUSIONS AND FUTURE WORK ....................... 153

Cycloalkanoporphyrins .................................. 153
5-NO2 OEP and its Divalent Metal Complexes ......... ....... 154
Porphyrin Photosensitizer ................. ............... 156

REFERENCES ......................................... 157

BIOGRAPHICAL SKETCH .............................. 168

. . 1 18

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



John D. Laycock

December 1994

Chairperson: Dr. Richard A. Yost
Major Department: Chemistry

The research reported in this dissertation employs tandem mass

spectrometry (MS/MS) to study a variety of topics in porphyrin chemistry. Closely

related structures of geoporphyrins containing exocyclic rings are shown to be

distinguishable by MS/MS. We show strong evidence that the non-planarity of 5-

NO2 octaethylporphyrin (OEP) and its divalent metal complexes in conjunction

with oxygen migration, induces unusual porphyrin fragmentations in the gas

phase. Finally, porphyrin photosensitizing reactions are studied by on-line and

off-line thermospray ionization (TSP) MS/MS.

The electron ionization tandem mass spectrometric (EIMS/MS) analysis of

22 cycloalkanoporphyrin (CAP) standards is presented. The daughter spectra of

molecular ions of 6 different skeletal types show that CAPs bearing different

isocyclic rings can be distinguished by the variation of the relative abundance of

three ions ([M-43] [M-44]+, and [M-45] ). When the normalized intensities of the

three ions are plotted on a ternary diagram, distinct clusters of points appear for

each of the skeletal types studied. This approach is applied to daughter spectra

of molecular ions from partially separated mixtures of geoporphyrin extracts and

the implications for biomarker analysis are discussed.

The El MS/MS and high resolution El' data for free-base 5-NO2 EP show

that the porphyrin macrocycle molecular ion cleaves a pyrrole unit in the gas

phase. This phenomenon is significant since ring scission has previously only

been observed for substituted porphyrins by surfaced-induced decomposition

during H2 chemical ionization MS. Daughter spectra for the molecular ions of

divalent metal complexes of 5-NO2 OEP show a metal-dependency in their

fragmentations. The most metal-dependent daughter ion, the [M-86] ion,

correlated well with metal- and conformation-sensitive infrared and Raman bands.

Oxygen migration and non-planarity are important factors in inducing both of

these fragmentations.

Porphyrin photosensitizing reactions of interest to photodynamic therapy

for cancer and their characterization by on-line and off-line photochemistry

TSP/MS/MS is discussed. The on-line monitoring of the photosensitization of

tryptophan by hematoporphyrin is demonstrated. The construction of an on-line

photochemistry cell and its evaluation by comparison to off-line photochemistry

experiments is discussed.


This dissertation can be divided into two sections (each describing the use

of tandem mass spectrometric techniques to study porphyrins) The first section

involves structural studies of geoporphyrin standards and synthetic

nitroporphyrins. The second involves the structural elucidation of photoproducts

of porphyrin photosensitizing reactions. Introductory topics that follow in this

chapter are intended to familiarize the reader with background and previous work

of relevance to research discussed throughout the dissertation. A brief

introduction to tandem mass spectrometric instrumentation and techniques is

given. Then a historical perspective of porphyrin research is presented, followed

by sections pertaining to the following areas of interest in porphyrin chemistry:

inorganic chemistry of porphyrins, biological chemistry of porphyrins, geological

chemistry of porphyrins, porphyrins of biomedical significance, and finally, the

mass spectrometry of porphyrins.

Background information that is pertinent to each individual chapter will be

covered therein. The dissertation will be summarized and put into perspective

with previous work at the end of this chapter (Scope of Dissertation).


Tandem Mass Spectrometry

All of the tandem mass spectrometric studies described within this

dissertation were performed using a triple quadrupole mass spectrometer

(TQMS),(Figure 1-1). The TQMS was first described by Yost and Enke in 1979.

The arrangement of the three quadrupoles (Q1, Q2, and Q3) allows for two

separate stages of mass analysis (MS/MS) [Yost and Enke, 1979; Yost and

Fetterolf, 1983]. The advantages that an additional stage of mass analysis afford

are numerous [Johnson and Yost, 1985]. They include: first, a reduction in the

chemical noise and thus decreased limits of detection, and second, minimized (or

eliminated) need for sample clean-up and/or chromatographic separation.

Figure 1-2a shows a TQMS in the MS operation mode. For single-stage

mass separation experiments, either Q1 or Q3 may be scanned. The other two

quadrupoles are operated in the radio frequency (RF) only mode, allowing

essentially all masses to pass. The two MS/MS scanning modes that were utilized

in this dissertation research were the daughter scan and the parent scan.

In the daughter scan experiment (Figure 1-2b), one ion of a particular

mass-to-charge ratio (m/z) of interest (the parent ion) is allowed to pass through

the first quadrupole (Q1). This ion is then passed through the second quadrupole

with a selected collision energy. Within Q2 (RF only), the selected ion undergoes

collisions with an inert target gas (typically 1-3 mtorr of nitrogen or argon). The

collision imparts internal energy to the analyte ion; if the energy is sufficient,





II I (
Iii i
I i II

I I I a
:, I i :'

o a


o a)


2 I-


o -r
Cl) I-





^--*^______________ __ i____
-------- ------
;--_------ --i-



(d) Q1 Q2 Q3


Figure 1-2: Scan modes of the triple quadrupole mass spectrometer: a) Q1 MS;
b) daughter scan; c) parent scan; d) selected reaction monitoring
SRM scan.






fragmentation of the ion, or collisionally induced dissociation (CID), occurs. The

resultant fragment ions (daughter ions) pass into the third quadrupole Q3 were

they can be mass analyzed. The spectrum thus obtained by scanning Q3 is

called a daughter spectrum. The daughter spectrum is particularly useful in the

structural elucidation of analyte ions.

In the parent scan (Figure 1-2c), a range of parent ions are allowed to pass

through Q1. These ions are then passed through the collision cell (Q2), where

CID occurs. The Q3 is set such that only one daughter ion of a particular m/z is

passed. Any ions which fragment to give a daughter ion of this m/z will be

detected and are referred to as parent ions. The parent spectrum is useful in

identifying ions which fragment to form a common substructure. The parent scan

is also useful in determining mechanisms of the reaction pathways of a given

parent ion.

The various MS/MS scanning modes allow for the development of unique

analytical schemes. For instance, selected reactions can be monitored (SRM) by

passing only one parent ion through Q1 and passing only selected daughter ions

through Q3 (Figure 1-2d). The chemical noise is significantly reduced in the SRM

mode and trace compounds can be detected in complex matrices [Johnson and

Yost, 1985; Johnson et al., 1986].

The SRM scheme may be also used to study ion/molecule reactions. By

replacing the inert collision gas of Q2 with a reactive gas, Q2 may be employed

as a reaction chamber. The scanning mode is essentially the same as the SRM

scan described above except that Q3 is scanned to allow reaction products to

pass. For instance, Freeman et al. [1990b] developed a method for reacting

model nucleophile or DNA base ions with electrophilic gases; Q3 was scanned

to detect adduct ions. The utilization of this scheme in order to mimic

DNA/carcinogen reactions in solution has been shown [Freeman et al., 1990b;

Annachino, 1993].


Porphyrins are cyclic tetrapyrroles that are of great importance to all living

cells; they are the active structural moiety of hemoglobin, myoglobin, all the

cytochromes, and the chlorophylls (dihydroporphyrins). The structure of

porphine, the simplest porphyrin, is shown in Figure 1-3. Four pyrrole units are

linked by four one-carbon bridges; the entire structure has a planar geometry due

to the conjugation of the sp2 carbons. The bridge carbons are termed the meso

positions. The pyrrolic carbons adjacent the nitrogen are a-positions and the

peripheral carbons are /-positions. The IUPAC numbering system is also shown

in Figure 1-3. The porphyrin may be substituted by a variety of functional groups

at either the meso or ft positions.


Figure 1-3: Porphine, the simplest porphyrin.

Historical Perspective

In 1844, Verdeil chemically altered chlorophyll into a red pigment and

suggested that a structural relationship exists between chlorophyll and heme

[Dolphin, 1978]. In 1915, Willstatter received the Nobel Prize for his purification

and structural determination ot plant pigments, especially that of the chlorophylls

[James, 1993]. About a decade later, Hans Fischer, widely regarded as the

"father" of modern porphyrin chemistry, was awarded a Nobel Prize for his

synthesis of hemin from dipyrromethanes. Fischer later reviewed the early work

of porphyrin structural and synthetic studies in his three volume work Die Chemie

des Pyrrols [Fischer and Orth 1937a.b: Fischer and Stern 1940]. In 1934 Alfred

Treibs, a colleague of Fischer's, identified vanadyl

deoxophylloerythroetioporphyrin (DPEP) as a major metalloporphyrin in

petroleums and Shale. He proposed ancient chlorophylls as the source of DPEP;

out of this original theory, the field of molecular organic geochemistry developed

(see Figure 1-6 and the section on the geochemistry of porphyrins).

In 1962 Kendrew and Perutz shared the Nobel Prize in chemistry for their

structural studies on the oxygen-binding proteins hemoglobin and myoglobin by

X-ray crystallography (Watson and Crick) shared the Nobel Prize in medicine

during the same year for their similar structural elucidation of DNA) [James, 1993].

Robert Woodward, considered by many the foremost organic chemist of the

twentieth century, was awarded the 1965 Nobel Prize for his total synthesis of the

tetrapyrrole vitamin B,2 [James, 1993].

In 1964, Falk published his book Porphyrins and Metalloporphyrins which

discussed and reviewed some of the physico-chemical aspects of porphyrins.

However, most of the porphyrin research until the mid 1970's dealt with structural,

synthetic and biosynthetic topics. More recent research focuses on the function

and mode of action of porphyrins in the various important biological functions.

Dolphin edited a seven-volume series covering a wide range of topics in the

chemistry and biochemistry of porphyrins in 1978.

Research interest in porphyrins has increased dramatically in recent years

as scientists are discovering a diverse array of reactions dependent on porphyrins

for their function. Much of the present porphyrin research is aimed at

understanding the fundamental processes of photosynthesis and catalysis.

Deisenhofer, Huber and Michel won a Nobel Prize in 1988 for their work probing

the sequence of events occurring in porphyrin chromophores utilizing magnetic

resonance techniques, laser spectroscopies, and X-ray crystallography

[Deisenhofer. 1989]. With in vivo functionality and electronic properties of

porphyrins better understood, in vitro biomimetic photocatalysts may be

eventually developed [Fajer, 1991]. Porphyrins are also of current interest in the

area of molecular electronics; Crossley and Burn [1991 ] have recently synthesized

a "wire" consisting of a linear array of porphyrin macrocycles.

Inorganic Chemistry of Porphyrins

Porphyrins chelate with virtually all metallic elements. The coordination of

a metal significantly alters the physical, chemical and spectroscopic properties of

the porphyrin. The free-base porphyrin H2(P) chelates with the metal (M) to form

the metalloporphyrin M(P) in what is termed the metallation reaction (Figure 1-4).

The simplest case, and the only one that will be dealt with here, is the formation

of a square-planar complex from a dipositive metal ion (M2") and a dinegative

porphyrin anion (P2). The reverse of this reaction is demetallation, and occurs in

the presence of acids (Figure 1-4).

Five different stability classes of metalloporphyrins have been distinguished

based on their ease of demetallation in the presence of various protic reagents

[Falk 1964; Buchler, 1975]. Buchler used a stability index to relate inherent

properties of the metal to the stability of the metalloporphyrin complex [Buchler,

1975; Buchler, 1978]. The Buchler stability index (S,) is easily calculated from

fundamental properties of the metal:

S= 100(PE)(V)/R

Where PE is the Pauling electronegativity of the metal, V is the valence of the

metal, and R is the ionic radius of the metal. The lower the stability index, the

greater the ease of demetallation of the porphyrin in solution.




Porphyrins can have a significant loss of planarity with the coordination of

certain metals. This is of interest because many biologically active porphyrins are


Biological Chemistry of Porphyrins

Cyclic tetrapyrroles are involved in many vital biological processes.

Electron and oxygen transport processes, photosynthetic reactions, oxygen

insertion, and CO, removal from tissues are all dependent on cyclic tetrapyrroles

[Addison et al., 1977]. Four important types of cyclic tetrapyrroles are porphyrins,

chlorins (dihydroporphyrins), bacteriochlorins (tetrahydroporphyrins), and corrins.

In Figure 1-5, the structure of a metalloporphyrin and a metallochlorin are

shown. In the chlorin. a peripheral double bond has been removed through

reduction; thus, two additional hydrogens are present at the /-carbons of the

pyrrole ring. The mere addition of these two hydrogens is responsible for a

substantial shift in the UV-Vis absorption peaks. Thus, while most porphyrins are

red in color, the chlorins are green.

Also shown in Figure 1-5 is the structure of protoheme (heme). Heme is

the iron (II) complex of protoporphyrin IX, and is the prosthetic group in

hemoglobin, myoglobin, the cytochromes, and many of the peroxidases and

catalases. The iron (II) ion is usually octahedrally coordinated, meaning that two

additional ligation sites remain available in the heme-porphyrin complex.

\\ /
\/ \ /



I -I


Chlorophyll a

Figure 1-5: Structures of porphyrins and chlorins.



Hemoglobin contains four protein-bound heme moieties, and is vital in the

transportation of oxygen in the bloodstream. The oxygen is carried by axial

(perpendicular to the ring) ligation to the iron. Once one molecule of oxygen is

bound, the other coordination sites of the hemoglobin have an increased affinity

for binding oxygen [Marks, 1969].

In deoxymyoglobin, one of the axial sites of the protein-bound heme

complex is taken up by a nitrogen of histidine. In myoglobin, an oxygen is

reversibly bound at the other axial position. This oxygen is only relinquished

when muscle oxygen demand is greater than the oxygen supply from the blood

(during strenuous exercise). In this way, myoglobin acts as an emergency store

of oxygen [Marks, 1969].

The cytochromes are responsible for the respiratory electron transport

processes occurring in the mitochondria and the chloroplasts. Cytochromes are

classified as a, b, or c depending on their characteristic absorption spectrum,

with cytochrome b, c, and c, having heme as its prosthetic group. The

cytochromes transfer electrons through reversible changes in the iron atom

between the reduced iron (II) and oxidized iron (111) states [Lemberg and Barrett,

1973; Ortiz de Montellano, 1986].

The structure of chlorophyll a is shown in Figure 1-5. The structure is a

chlorin, as one of the peripheral double bonds is absent (thus the green color).

The coordinated metal is magnesium. The chlorophylls are responsible for the

photosynthetic reactions that harvest the sun's energy and store it as chemical


potential energy. Bacteriochlorophylls constitute the light-harvesting antenules of

photosynthetic bacteria [Deisenhofer and Michel. 1989; Tronrud etal., 1986]. The

bacteria funnel photons into a complex called the reaction center; a separation

of oppositely charged ions across a membrane creates a potential gradient. The

stored electrical energy is the driving force of the biochemistry of the organism

[Norris and Schiffer, 1990: Feher et al., 1989].

Vitimin B,, a corrin, is a cyclic tetrapyrrole structurally related to heme, but

with a cobalt (II) ion as the coordinated metal. Vitimin B,, is an essential nutrient

and is present in coenzymes responsible for a number of reactions, including the

oxidation of fatty acids and the synthesis of DNA [Addison et a., 1977].

In many enzymes a metalloporphyrin is the biochemically active site.

Porphyrins are of great interest in the development of new catalysts; much

research has been devoted to the study of porphyrin-initiated reactions. Some

porphyrins are known to be strong photosensitizing agents. Because of these

photosensitizing properties, porphyrins have found utility as photodynamic

therapy (PDT) agents in the treatment of cancer [Doiron and Gomer, 1983].

Details on PDT are covered in detail in Chapter 4 of this dissertation.

Geological Chemistry of Porphyrins

The inherent stability of the porphyrin macrocycle coupled with the

abundance of porphyrinic structures in living systems makes them important to


geochemistry. Treibs [1934; 1936] hypothesized that geoporphyrins derived from

the chlorophyll in ancient plants undergo decarboxylation, reduction, dealkylation,

and oxidation. Figure 1-6 shows the series of reactions occurring in the

diagenesis scheme proposed by Treibs [1934; 1936]. The color of each

compound and its nominal molecular weight is shown as these compounds have

been classified by UV-Vis spectroscopy [Stern and Wenderlein, 1936] and by

mass spectrometry [Baker et al., 1968; Baker and Smith, 1973].

The first reactions involve the demetallation, saponification, and

decarbomethoxylation of chlorophyll a; Treibs proposed thatthese reactions occur

at the water/sediment interface with little external stress. The subsequent

reactions (reduction, aromatization, decarboxylation, and chelation) are said to

occur as the sediments are compacted and the thermal stress increases. Treibs

explained that the order of these reactions are interchangeable and are

dependent on the depositional environment.

Porphyrins resulting from these reactions are ubiquitous in petroleum

deposits, yet comprise only a small portion of the total carbon. There are a

variety of peripheral substituents present (meso substitutions generally do not

occur except for isocyclic rings) making the geoporphyrins important as

biomarkers [Eglinton and Calvin, 1967].

Biomarkers are defined as compounds which retain a basic carbon

skeleton during and after deposition in the earth's sediments. The biomarkers

can be related to a specific source of organic materials and for this reason are


S- 0

z z z


o -

35 C _; E -


> o
tZ -

zo z o

z z -= 0Z
xE ~U)


sometimes referred to as chemical fossils [Bonnett et al., 1991]. Geoporphyrins

have proven useful as biomarkers because their structural complexity allows for

fingerprinting of samples for oil-oil or oil-source rock correlations [Corwin, 1960;

Callot et al., 1987; Chicarelli et al., 1987; Filby and Van Berkel, 1987]. Current

research aims at clarifying the evolution of biological precursors into geoporphyrin

products. That extended isocyclic rings (up to seven carbons) and

benzoporphyrins are present in geoporphyrin extracts suggests that some

processes are unaccounted for under Treibs' digenesis schemes [Bonnett et al.,

1991]. The Treibs scheme also does not account for the occurrence of high

carbon-number porphyrins observed by Johnson et al. [1985] by tandem mass

spectrometry. Some of the previous porphyrin work in our research group has

been aimed at obtaining structural information on geoporphyrins using tandem

mass spectrometry [Quirke etal., 1988; Beato etal., 1989a,b; Quirke etal., 1989].

Porphyrins of Biomedical Importance

Porphyrins are of great importance to many areas of the biomedical and

pharmaceutical sciences [Doiron and Gomer, 1984; Bonnet et al., 1989; Gomer,

1990; Morris, 1991: Kessel et al., 1991], especially those involving

photoirradiation. It has long been known that certain porphyrins cause

photosensitivity in man. The photosensitivity associated with malfunctions of


porphyrin metabolism, such as in the porphirias, is attributed to the presence of

photoactive porphyrins under the skin [Blum, 1941].

The use of photosensitizing porphyrins in the treatment of malignant tumors

is becoming increasingly important. Thousands of cancer patients have been

treated with Photofrin I, a commercial product that is employed for photodynamic

therapy (PDT) [Gomer, 1990]. Photofrin consists of a complex mixture of

porphyrin polymers derived from hematoporphyrin. One of the major current

research efforts centers on characterizing the Photofrin mixture in order to

determine which of the its components are the active PDT agents [Bonnett et al.,

1981; Berenbaum et al., 1982; Dougherty, 1984]. Another well-researched area

involves the development of a new generation of synthetic cationic and anionic

water-soluble porphyrin PDT agents [Le Nouen et al., 1989; Pasternack et al.,

1985]. Despite considerable research efforts in these areas, the modes of action

of the porphyrins are not well known. It is uncertain how cell components (and

which of them) sustain enough damage to cause cell death.

Mass Spectrometry of Porphyrins

Mass spectrometry has been shown to be a powerful tool for the structure

elucidation and identification of porphyrins. Past research in our group has been

involved with the detection and structure elucidation of high-carbon number

geoporphyrins [Johnson et al.. 1986; Cuesta et al., 1989] and with the


investigation of porphyrin related phenomena. Tandem mass spectrometry of

doubly charged ions and the surface-induced decomposition of porphyrins in

chemical ionization are two areas that have been well-researched [Beato et al.,

1989a; Beato et al., 1991].

Electron Ionization (El)

A compound that serves as a model for the fragmentation of biological and

geochemical porphyrins is octaethylporphyrin (OEP). The full-scan El mass

spectrum of OEP is shown in Figure 1-7a. The ring structure remains intact as

evidenced by the absence of mono-, di-, or triprollic ions in the El mass spectrum.

The conjugation and high stability of the ring prevent the loss of pyrollic units.

In Figure 1-7b, the OEP mass spectrum has been blown up by a factor of five and

expanded to emphasize the singly charged region of the mass spectrum. The

spectrum shows that eight successive losses of 15 mass units occur from the

ions. Seibl first reported in 1968 that the fragmentation of the peripheral ethyl

substituents of OEP occurs via a benzylic type #-cleavage in which successive

losses of methyl radicals from the molecular ion are observed (Figure 1-8).

Regardless of the length of the alkyl chain, the fragmentation of alkyl-substituted

porphyrins is of the 3 type [Britton, 1985; Johnson et al., 1986]. The bond

between the first and second carbons is homolytically cleaved to form an alkyl

o *o




0 (n
-r a

() -r





o a

C c
o z

, cz


(A) a
ca a)
c" EI
N E a) .
'E a)3
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g qcr




+ _

n ----aiM^^



',0 -
i I


S'4 -







I i i fi I i i






2 E


U U)

N 0r

* 0

ir a.
____ w




radical. This phenomenon has proven useful for the determination of the

peripheral substituents of porphyrins.

There is a high abundance of doubly charged ions in the mass spectra of

porphyrins (Figure 1-7a); this can be attributed to the large number of r electrons
that are readily available [Jackson et al., 1965]. Nuclear magnetic resonance

(NMR) data show that the charges are separated by localization at two of the

central nitrogens [Chakraborty et al., 1982]. The l-cleavage fragmentation

occurring in the doubly charged region of the mass spectrum is significantly

greater than is observed in the singly charged region. Additionally, some a-

cleavage occurs in the doubly charged region [Beato et al., 1989b].

There is a marked influence on the fragmentation that is dependent on the

chelated metal ion. The relative abundance of the ions in the doubly charged

region of the mass spectrum has been shown to correlate with the modified

Buchler stability index (S,) for the metal, while the singly charged region remains

relatively unaffected by the nature of the metal [Beato et al., 1989b].

Chemical Ionization (Cl)

Chemical ionization with methane as the reagent gas has proven useful for

the determination of porphyrin molecular weights [Eglinton etal., 1979]; however,

the most interesting phenomenon occurring in chemical ionization of porphyrins

is surface-induced decomposition in the presence of H2 or NH3 [Beato et al.,

1991]. Porphyrin molecules that have deposited on the inside of the ion volume


are reduced and then subsequently revaporized as a result of radiative heating

by the direct exposure probe. As a result of the reduction to porphyrinogen (a

hexahydroporphyrin), the bridge carbons are now sp3 hybridized; thus they are

no longer conjugated. This acid-labile macrocycle fragments to give mono, di,

and tripyrrole fragments which yield structural information with regards to pyrrole

sequence and individual pyrrole composition [Beato et al., 1991; Van Berkel etal.

1989a.b; Shaw et al., 1981].

Tandem Mass Spectrometry (MS/MS)

Tandem mass spectrometry has been utilized to gain structural information

from the molecular ions of various porphyrins and especially those of

geoporphyrins [Johnson et al., 1986]. Partially separated mixtures of

geoporphyrins can be analyzed by taking daughter spectra of the molecular ions

of interest. Structural information on these porphyrins is thus obtained. By

performing MS/MS, the nature of the peripheral substituents may be clarified.

Tandem mass spectrometry of doubly charged porphyrin ions is of interest

since porphyrins are one of the few classes of compounds (small peptides and

polycyclic aromatic hydrocarbons are others) whose ions retain a double charge

when fragmented via CID [Appling et al., 1983; Hanner et al., 1982]. Most

compounds with doubly charged ions would retain only a single charge after CID

owing to the charge exchange reactions that occur in the collision cell (Q2).


Doubly charged porphyrin ions are different in this respect because they are

resonance-stabilized and maintain their double charge during CID [Beato et al.,


Scope of Dissertation

The dissertation is divided into two sections based on the methods

employed and the analytical applicability of the work presented. Chapters 2 and

3 concentrate on the study of ion fragmentation pathways of series of porphyrin

standards in the gas phase. Chapter 4 is a study of the aqueous-phase

photosensitizing reactions of porphyrins with a biological substrate. Chapter 5

summarizes the dissertation and outlines areas of future work.

In Chapter 2, the electron ionization tandem mass spectrometric

(EI/MS/MS) analysis of cycloalkanoporphyrin (CAP) standards is presented. First,

a background of bi -marker analysis and a survey of current research in this area

is discuss d. The structure of the skeletal types studied and th r possible

relationship to naturally occurring geoporphyrins is shown.

The daughter spectra of the molecular ions of 6 different skeletal types are

presented. The data show that the CAPs bearing different isocyclic rings can be

distinguished by the variation of the relative abundances of three ions ([M-43]*,

[M-44], and [M-45]*) within each compound's spectrum. When the three ion's

normalized intensities are plotted on a ternary diagram, distinct clusters of points

appear for each of the skeletal types studied. For comparison, the technique is

applied to daughter spectra of molecular ions from partially separated mixtures

of geoporphyrin extracts. The implications of this work towards porphyrin

biomarker analysis is discussed.

In chapter 3, meso-nitro octaethylporphyrin (5-NO2 OEP) and its divalent

metal complexes are studied by electron ionization tandem mass spectrometry

(EI/MS/MS) and by high resolution El mass spectrometry. The reason 5-NO2 OEP

was chosen is that has been studied extensively by various optical spectroscopic

techniques. The structure is known to be non-planar; indeed, 5-NO2 OEP has

been shown to be useful as a model compound for cytochrome-c [Shelnutt et al.,


The data for the free base 5-NO2 OEP show that the porphyrin macrocycle

molecular ion cleaves a pyrrole unit in the gas phase. This phenomenon is

significant since ring scission has only previously occurred for substituted

porphyrins by surfaced-induced decomposition [Beato et al., 1991]. The results

of isotopic labelling with 13C and 15N and high resolution mass spectrometry

confirm ion compositions deduced from MS/MS spectra of unlabelled 5-NO2 OEP.

The migration of oxygen occurs in an unusual multi-step fragmentation pathway

in which several bonds are broken.

Divalent metal complexes of 5-NO2 OEP are also analyzed by MS/MS. A

daughter spectrum is obtained for the molecular ions of each of the various

metalloporphyrins. The fragmentations are discussed and compared to those

observed for the free-base 5-NO, OEP. Trends within the series of metals studied

are discussed and are related to other metal-dependent spectroscopic properties.

In Chapter 4, photosensitizing reactions of porphyrins and their

characterization by off-line and on-line photochemistry/thermospray ionization

tandem mass spectrometry is discussed. The intended application of this work

is to the study of the reactions of photodynamic therapy cancer treatment agents.

The chapter begins with an introduction to photosensitizing reactions and

goes on to discuss the utilization of these photosensitizers as therapeutic cancer

treatment agents. Some of the techniques for studying on-line solution phase

reactions as well as photochemical reactions will be described.

Finally, the development of our instrumentation and methodology is

discussed. Comparisons are drawn between our system and commercially

available components and between the methods of on-line and off-line

experiments. The results of the photolysis experiments are discussed and put

into perspective with known biological redox reaction pathways.

Chapter 5 summarizes the work presented in the dissertation. Areas of

possible future work are discussed for each of the research topics discussed in

the chapters.



The electron ionization tandem r iass spectrometric analyses of six skeletal

types of cycloalkanoporphyrins (CAP's) together with their Ni(ll) and VO(II)

complexes are presented. Analysis of the daughter ion spectra of the molecular

ions (M') indicates that it is possible to distinguish isomers with different sizes

of isocyclic rings. For instance, deoxophylloerythroetio, DPEP, porphyrins with

a 5-membered isocyclic ring show a characteristic, intense [M-44]+ daughter ion,

whereas CAP-6 porphyrins (CAPs bearing a 6-membered isocyclic ring) display

an intense [M-45]+ daughter ion. As a result of these findings, it is now possible

to identify the skeletal type of CAP porphyrins in intact or partially separated

geoporphyrin mixtures. Data from previous studies on the porphyrins of New

Albany Shale (Mississippian-Devonian, Indiana USA) are used to demonstrate the

potential applications of this approach.

Geoporphvrin Skeletal Types

Geoporphyrins (geologically-occurring porphyrins) occur mainly as

complicated mixtures of Ni(ll) or VO(II) complexes in sediments, oil Shales,

bitumens, coals, phosphorites and crude oils [Treibs, 1934]. At least ten different

skeletal types of geoporphyrins are known [Chicarelli et al., 1987; Filby and Van

Berkel, 1987; Ocampo et al., 1987; Callot et al, 1990; Keely et al., 1990]. In

Figure 2-1 the structural types studied here are shown; for the remainder of this

chapter the reader will be referred to specific structures within Figure 2-1 by an

underlined numeral. Two skeletal types are believed to occur most commonly-

the ETIO (1) and the deoxophylloerythroetio (DPEP) (2). The term, CAP-n

porphyrin, will be used to describe cycloalkanoporphyrins bearing a single

isocyclic ring with n-carbons (i.e., the DPEP is a CAP-5 porphyrin). Similarly,

CAP-N-Me indicates that there is a methyl substituent on the isocyclic ring. Four

different skeletal types of CAP-porphyrin have been isolated from geological

samples (2,3,4,6: R9 = CH,). The tetrahydrobenzoporphyrins (THB, Z) themselves

have not been reported; however, the di-CAP analogues (8a,b) and

benzoporphyrins have been identified [Kaur et al., 1986; Verne-Mismer et al.,

1987]. Thus, the THB skeleton may also occur (as might the des-methyl

CAP-6, 5).

The geoporphyrins are a potentially valuable compound class for the

fingerprinting of oils in oil-oil and oil-source rock correlation studies in oil


(a) RI,R2,R5,R8 = CH3
R2,R4,R7 = C2H5
(h) RI,R4,R5,R8 = CH,
R2,R3,R7 = CH5
(C) RI,R2,R3,R5,R8 = CH3
R4,R7 = C2H5


(a) RI,R2,R5,R8,R9 = CH3
R2,R4, = C2H, ;R6 = H
(h ) RI,R2,R3,R5,R8 = CH3
R4,R6 = CH ;R, = H

Figure 2-1: Molecular structures of cycloalkanoporphyrin (CAP) standards.





(a) RI,R2,R3,R5,R8 = CH3
R4,R7 = C2H,
(b) RI.R4,R5,R8= CH
R2,R3, = CH,; R7 = H
(c) RI,R4,R5,R8 = CH3
R2,R3,R7 = CH,



(.) R7=H
(2) R7=CHA

Figure 2-1 -- continued.

exploration [Beato et al., 1989a; Concha et al., 1991]. Similarly, they may prove

useful for the identification of oils and tar balls from oil spills [Johnson et al.,

1986]. In order to fully realize their potential for such applications, the

components of the mixtures must be separated, characterized and quantitated

precisely and efficiently.

Analysis of Geoporphyrins

Recently, significant advances have been made in both gas

chromatographic and high performance liquid chromatographic separations of the

metal-free, nickel and vanadyl geoporphyrin mixtures [Sundararaman, 1985;

Barwise et al., 1986; Chicarelli et al., 1986; Callot et al., 1990; Peng et a., 1992].

The Bristol [Chicarelli et al., 1987] and Strasbourg [Ocampo et al, 1987; Callot

et al., 1987] groups have provided a valuable database of geoporphyrin structures

as a result of their elegant structure elucidation studies. The work of the Freeman

group on the identification of geoporphyrins using visible spectrophotometry

provides intriguing possibilities for rapid characterization [Freeman and Haver,

1990; Freeman et al., 1990a]. High performance liquid chromatography/mass

spectrometry (LC/MS) also remains a technique of considerable potential [Bonnet

et al., 1991]. In particular, the development of LC/MS using electrospray

ionization provides an excellent method for the quantitation of geoporphyrin

mixtures [Van Berkel et al., 1993]. Nevertheless, the presence of a range of


isomeric CAP porphyrins in geoporphyrin mixtures provides a formidable barrier

to complete analysis of such samples.

The CAP porphyrins may be distinguished by 'H NMR [Callot et al., 1987;

Ocampo et al., 1987]; however, this requires the isolation of individual compounds

in substantial quantities (typically >100 p[g). This is entirely unsuitable for routine

mixture analysis. Clearly, the best hope for success would seem to lie in either

GC/MS or LC/MS analyses of the mixtures, but there are no previous reports that

it is possible to distinguish and assign isomeric CAP porphyrins by mass


Previous research in our group has demonstrated that electron ionization

tandem mass spectrometry (EIMS/MS) is a powerful analytical tool for

characterization of geoporphyrin mixtures [Johnson et al., 1986; Quirke et al.,

1989; Beato et al., 1991; Concha et al., 1991 ]. In addition, we reported that many

of the CAP geoporphyrins isolated displayed an abundant, unexpected [M-44]+

ion in the daughter ion spectra of their molecular ions, M+t [Quirke et al., 1989;

Beato et al., 1991]. At that time, however, it was impossible to determine whether

the isomeric CAP porphyrins could be distinguished by such spectra because

pure standards were unavailable.

In this chapter, the EIMS/MS daughter ion spectra of the molecular ions of

each skeletal type of CAP geoporphyrin are presented. Then the implications of

the data for analysis of geoporphyrin mixtures are discussed.




The Ni(ll) and VO(II) complexes of the CAP-5 porphyrins 2a, 3 were gifts

from Dr. R. Ocampo (Universite Louis Pasteur, Strasbourg). The CAP-6

porphyrins 4a,b, 5a and the CAP-7 porphyrin 6 were gifts from Dr. P. S. Clezy

(University of New South Wales, Sidney) The CAP-5 porphyrin 2b, the CAP-6

porphyrins 5b,c and the THB porphyrin 7 were gifts from Dr. T. D. Lash (Illinois

State University, Normal). The C,3 CAP-5 porphyrin 2c was isolated from the

bitumen Gilsonite (Eocene, Uinta Basin, Utah, USA) using the method of Quirke

and Maxwell [1980]. The procedures for the isolation and initial characterization

of the porphyrins from the New Albany Shale (Mississippian-Devonian, Indiana

USA) have been described previously [Beato et al., 1991].


All mass spectra were obtained using a Finnigan TSQ 45 triple quadrupole

tandem mass spectrometer equipped with an INCOS data system. For EIMS, the

mass spectrometer was tuned using perfluorotributylamine (FC43). Tuning was

not deemed to be complete until there was complete resolution of the ions m/z

502 and 503. EIMS spectra were obtained using a direct exposure probe (DEP).


The porphyrin in dichloromethane (DCM) solution was placed on the DEP filament

and allowed to evaporate. The DEP was then heated from ambient to 600C at

600C/min. The El emmision filament current was 0.3 mA with an electron energy

of 70 eV. The spectra were obtained by scanning the first quadrupole, Q1, from

m/z 150 m/z 800 at a rate of 0.8 s. In ElMS/MS mode, the instrument was tuned

using Ag(ll) 5-nitro-2,3,7,8,12,13,17,18-octaethylporphyrin. We have discovered

this porphyrin to be particularly valuable for tuning because the [M-75]+ ion is by

far the most abundant daughter ion of the M' ion; the typical fragmentation

pathways by /-cleavage of the ethyl substituents are not observed (see Chapter

3 of this dissertation). The sample was volatilized using the DEP as described

previously. The porphyrins were ionized with an electron energy of 70 eV and an

emission current of 0.3 mA. The daughter ion spectra were obtained using argon

(1.6 mtorr) for collisionally activated dissociation in the second quadrupole with

collision energies ranging from 10 eV to 28.5 eV.

Metal Insertion

Nickel porphyrins were prepared by treatment of the metal-free porphyrin

with excess Ni(ll) acetate in refluxing acetic acid as described by Buchler [1975].

Vanadyl porphyrins were prepared by refluxing the porphyrin with vanadyl sulfate

in either acetic acid/pyridine (2:1 v:v) as described by Stanley et al. [1991] or in

N,N-dimethylformamide as described by Adler et al. [1970].

Results and Discussion

Where possible the metal-free, Ni(ll) and VO(II) complexes of each CAP

porphyrin were studied by EIMS/MS; however, sometimes there was insufficient

sample to prepare the three compounds. The daughter ion spectra of the

molecular ions, M', of all the metal-free, Ni(ll) and VO(II) complexes of the CAP

porphyrins are summarized in Table 2-1. The daughter ion spectral data for

selected porphyrins, CAP-5 [Ni(ll) 2a], CAP-5-Me [VO(II) 3] CAP-6 (metal-free 5a

CAP-6-Me (metal-free 4b) CAP-7 [Ni(ll) 6] and THB [VO(II) 7] are shown in Figure


Previous EIMS/MS analyses of Ni(ll) and VO(II) complexes and the

corresponding demetallated geoporphyrins from the New Albany Shale

(Mississippian-Devonian, New Albany, Indiana, USA), and demetallated VO(ll)

porphyrins from Boscan oil (Cretaceous, W. Venezuela) revealed that the

daughter ion spectra of the molecular ions were quite reproducible over long time

periods (ten years) and with different operators of the instrument [Johnson et al.,

1986; Quirke et al., 1989; Beato et al., 1991; Concha et al., 1991]. Nevertheless,

it was essential to confirm the reproducibility of the method using CAP-standards.

Therefore, the EIMS/MS daughter ion spectra of the molecular ions of the Ni(ll),

VO(II) and metal-free CAP-6-Me (4a) and CAP-7 (6) were obtained at least four

times over a two-year period. The daughter spectra of the M' ion of the

compounds were quite reproducible. In particular, the pattern of daughter ions

Table 2-1. EIMS/MS Daughter Ion Spectra of the Molecular Ions of CAP


% Abundance (Relative to M-15 =100%)'
M-15 M-29 M-30 M-31 M-43 M-44 M-45 M-59

1 19
S 16
10 44
9 9

- 87
- 26
- 62


Ni2a C5
VO2a C5
Ni2b C5
FB2b C5
FBc2 C5

Ni3 C5M
V03 C5M

Ni4a C6M
VO4a C6M
FB4a C6M

Ni4b C6
VO4b C6
FB4b C6

FB5a C6
FB5b C6
FB5c C6

Ni6 C7
V06 C7
FB6 C7












1 13

S 28
4 41

1 9

2 5
S 6
1 9

4 7

'The ions were renormalized to [M-15] = 100% to clarify the correlation. In the
"M-X" columns, ions of less than 1% relative abundance are not listed.
FB = Free base; C5, C6, C7 = CAP-5, CAP-6, CAP-7 porphyrins respectively;
C5M, C6M = CAP-5-Me; CAP-6-Me respectively. Refer to Figure 2-1 for structures
of molecules.

518 100 2
527 100 4

Figure 2-2: Selected EIMS/MS daughter ion spectra of the molecular ions of
CAP porphyrin standards: a) Ni(ll) C32 CAP-5 2a (m/z 532); b) VO(II)
C31 CAP-5-Me 3 (m/z 527); c) CAP-6 5a (m/z 476); d) CAP-6-Me 4a
(m/z 476); e) Ni(ll) CAP-7 6 (m/z 532); f) VO(II) THB 7 (m/z 555).


J 59.0


* ase


t08.8;01 f)

440 4A

526 548


in the [M-43]* to [M-45] region of the spectrum varied less than 10% in

relative abundance.

All of the daughter ion spectra show [M-15]+ and [M-30]+ daughter ions,

which are formed by the classic P-cleavage of ethyl substituents of the
macrocycle. CAP porphyrins with different isocyclic rings produced either

different daughter ions or different distributions of daughter ions at [M-43]+,

[M-44] and [M-45]*. A ternary plot of the relative abundances of the three ions

for the 22 compounds listed in Table 2-1 demonstrates graphically that the CAP

porphyrin skeletal types can be distinguished using these three ions (Figure 2-3).

For this purpose, the sum of the relative abundances of the [M-43]+, [M-44]+ and

[M-45]+ ions was set to equal 100%. The differences for the CAP-5, CAP-6,

CAP-7 and THB porphyrins will now be discussed in turn.

Ni(ll), VO(ll) and metal-free CAP-5 porphyrins (2) all show [M-44]+ as either

the sole daughter ion or the dominant daughter ion in the [M-43]+ to [M-45]+

region of the spectrum The [M-45]' ion may also occur as a minor daughter ion,

with the highest relative abundance occurring for the Ni(ll) compounds (Table 2-1;

Figure 2-2). All of the five CAP-5 porphyrins studied clustered together in the

ternary diagram (Figure 2-3). Varying the substitution pattern, carbon number

and the nature of the chelated metal ion (if any) of the CAP-5 porphyrins

produced only minor variations in the daughter ion spectra of the M+ ion.

Nevertheless, the [M-44]' was always the dominant ion, and the [M-43]+ ion was









+ C

+ Cl

+ Ci


o .

) a,



a) a



1- 0







01 nc(3c>f



not detected (Table 2-1). Furthermore, the [M-441+ ion was always more abundant

than the [M-30]+ ion.

Only one CAP-5 porphyrin with a methyl substituent on the isocyclic ring

was studied (3). The daughter ion spectra of the M" ion of both the Ni(ll) and

VO(II) complexes of 3 revealed [M-44]' as the dominant ion in the [M-43]+ to

[M-45]' region of the spectra (Figure 2-2, Table 2-1). These compounds

clustered with the CAP-5 porphyrins in the ternary plot (Figure 2-3).

The Ni(ll), VO(ll) and metal-free CAP-6 porphyrins 4b, 5a, 5b and 5c show

[M-45]* as the dominant daughter ion in this region of the EIMS/MS daughter ion

spectrum. In contrast to the CAP-5 porphyrins, the fragmentation pattern can

vary more significantly with the nature of the chelated metal ion, the substitution

pattern, and the carbon number. The principal variations in these daughter ion

spectra lie in the relative abundances of the [M-45] and [M-30]1 ions and the

presence or absence of [M-44]+ as a minor daughter ion (Table 2-1). For

example, the daughter ion spectrum of the metal-free porphyrin 5a shows [M-30]

to be more abundant than [M-45], which is the reverse of what is observed for

both the Ni(ll) and VO(ll) complexes (Table 2-1). Despite these variations, the

CAP-6 porphyrins clustered together quite well on the ternary plot (Figure 2-3).

The presence of a methyl substituent on the 6-membered isocyclic ring

produces a significantly different fragmentation pattern in the [M-43] to [M-45]+

region of the spectrum. For Ni(ll), VO(ll) and metal-free 4b, the [M-44]+ and

[M-45]* daughter ions occur in similar relative abundances (Table 2-1; Figure

2-2). The three compounds formed a cluster between those of the CAP-5 and

CAP-6 porphyrins (Figure 2-3)

Only one CAP-7 porphyrin was studied (6). For the Ni(ll), VO(lI) and metal-

free forms the daughter ions [M-43]+, [M-44] and [M-45]1 were all observed in

relative abundance order of [M-43]' > [M-45] >- [M-44]+. The ions in this

region were less abundant than the [M-30]+ daughter ion for the metal

complexes, but were of similar abundance for the metal-free complexes (Table

2-1; Figure 2-2). The three porphyrins formed a cluster that was well separated

from those of the other porphyrin skeletal types (Figure 2-3).

The Ni(ll), VO(II) and metal-free forms of the THB porphyrin (7) all show

[M-43] as the dominant ion in this part of the EIMS/MS daughter ion spectrum.

For the metal-free porphyrin, this daughter ion is of lower relative abundance than

the [M-30] ion, which is the reverse of what is observed for the metal complexes

of 7 (Table 2-1; Figure 2-2). The three THB porphyrins formed a cluster that was

quite distinct from those of the other CAP porphyrins.

The THB porphyrins were similar to the CAP-7 porphyrins in that the [M-

43]+ ion appeared as the most intense ion of the [M-43]+, [M-44], and [M-45]+

cluster. In THB, there is a six-membered cycloalkyl group on the pyrrole ring; the

CAP-7 porpphyrin has the ring attached at the meso and f# carbons of the

porphyrin (Figure 2-1). In both of these cases, the dominant [M-43]+ ion could

correspond to the loss of CAH. The [M-43] could be the result result of

a-cleavage at one end of the exocyclic ring and fl-cleavage at the other with the

transfer of a single hydrogen.

In addition to the daughter ions already discussed, two other daughter ions

are often observed, [M-31] and [M-59]. The [M-31]+ ion was detected in many

of the spectra. Typically, it occurred in higher relative abundance in the spectra

of the CAP-6 porphyrin series than in the other skeletal types; however, it was

most abundant in the spectrum of the C31 CAP, 2c. At this point it does not

appear likely that this daughter ion will be valuable for distinguishing the different

types of CAP porphyrins because there is no obvious relationship between the

porphyrin skeletal types and its relative abundance. The [M-59] ion is of rather

more interest. It was observed in the daughter ion spectra of the molecular ions

of all the porphyrins studied except for the metal complexes of the THB porphyrin,

7. It was of similar relative abundance to the [M-43]+ ion for the CAP-7

porphyrins (Table 2-1). It was of lower relative abundance for the CAP-5

porphyrins except for the metal-free form of 2b. There was no clear pattern

between the relative abundances of the [M-59] and the [M-45] ions for CAP-6


In summary, the data indicate that careful analysis of EIMS/MS daughter

ion spectra of molecular ions permits the assignment of the isocyclic ring. The

size of the isocyclic ring may be ascertained using the following diagnostic


(a) CAP-5, 2, and CAP-5-Me porphyrins, 3, display a dominant [M-44]+ ion in
[M-43]+ to [M-451] region of the spectrum.
(b) CAP-5 and CAP-5-Me porphyrins are not distinguished by EIMS/MS.
(c) CAP-6 porphyrins, 5, are characterized by a dominant [M-45] in the this
region of the spectrum.
(d) The presence of [M-45]+ and [M-44] ions in similar relative abundance is
indicative of CAP-6-Me porphyrins, 4.
(e) The CAP-7 porphyrins show the following pattern of daughter ions: [M-43]f
> [M-45]+ > [M-44]+.
(f) THB porphyrins are characterized by a dominant [M-431+ ion.

Comparison With the Daughter Ion Spectra of
Molecular Ions of Geoporphvrins

It is appropriate to apply the results from the present study to the analysis

of geoporphyrin mixtures. Thus, the daughter ion spectra of the M+ ions of nine

geoporphyrins isolated from a total organic extract (bitumen-1) of the New Albany

Shale were re-examined [Beato et al., 1991]. The relative intensities of the

[M-43] [M-44] and [M-45] daughter ions are summarized in Table 2-2 together

with the re-normalized intensities, which were used for a ternary plot of the three

ions (Figure 2-4) Daughter ion spectra of the Ni(ll) C3, Ni(ll) C33, Ni(ll) C, and

VO(II) C32 CAP porphyrins together with two metal-free C30 CAP porphyrins are

shown in Figure 2-5. The selection criteria and the salient features of the

ElMS/MS analysis of the CAP geoporphyrins are discussed below.

The daughter ion spectra of the M' ions of Ni(ll) and VO(II) C30 and C32

CAP porphyrins were selected because the spectra are typical of those observed

for CAP geoporphyrins. The spectra of the demetallated C30 CAP porphyrins were

studied because they are isomeric, and therefore might be of different skeletal


Table 2-2. Analysis of the [M-43]+ to [M-45]+ Region of the Daughter Ion
Spectra of the Molecular Ions of Selected CAP Geoporphyrins of
New Albany Shale Total Organic Extract [Beato et al., 1991].

M.W. % Relative Abundance'
M-43 M-44 M-45

S 42
2 39

M-43 M-44 M-45

S 93
5 93





SThe ions were renormalized to [M-15]+ = 100% for comparison with the data in
Table 2-1.
** Re-normalized so that 1[M-43]+ + [M-44]+ + [M-45]+ = 100% for ternary plot
(Figure 2-4)
gThese isomeric CAP porphyrins were obtained by demetallation of the total
Ni(llnn) porphyrin mixture followed by thin layer chromatography.
FB = Free base; L = Less polar isomer; M = More polar isomer.

448 10
448 1

0) c-
+ a


0 n

,. C


c .0
+ >



m -

40 E
'cn4- 0


jO E

E cq


a) -
E So








Figure 2-5: Selected EIMS/MS daughter ion spectra of the molecular ions of
CAP geoporphyrins: a) Ni(ll) C30 CAP (m/z 504); b) VO(II) C32 CAP-
5-Me (m/z 541); c) Ni(ll) C33 CAP (m/z 546); d) Ni(ll) C, CAP (m/z
560); e) Less polar metal free C30 CAP isomer (m/z 448); f) more
polar C30 CAP isomer (m/z 448) from New Albany bitumen-l.

448 460 480









types. The spectra of the Ni(ll) and VO(II) C33 CAP porphyrins were very different,

and clearly warrented re-investigation. The Ni(ll) C34 CAP porphyrin was analyzed

because it is an example of a high carbon-number geopophryin porphyrin

[Johnson et al., 1986], which could not have been readily derived from

chlorophyll a via a Treibs' type degradation pathway [Treibs, 1936].

The VO(II) and Ni(ll) C3o and C32 CAP porphyrins displayed abundant

[M-44] ions (Figure 2-5, Table 2-2), indicating that were CAP-5 or possibly CAP-

5-Me porphyrins. On the ternary diagram (Figure 2-4), they formed a cluster in

the same place as the CAP-5 and CAP-5-Me standards. This is not surprising

because it has long been assumed that the CAP-5 porphyrins (DPEP) are usually

the dominant skeletal type of CAP porphyrins.

The presence of the dominant [M-44]+ ion in the more polar metal free C30

CAP isomer also was indicative of a CAP-5 porphyrin. In contrast, the [M-45]+

daughter ion was dominant in the less polar isomer, which indicates that it is a

CAP-6 compound (Figure 2-5, Table 2-2). On the ternary diagram, the less polar

isomer lay in the domain of the CAP-6 porphyrins. The more polar isomer lay

close to the CAP-5 porphyrins (Figure 2-4).

The daughter ion spectra of the Ni(ll) and VO(II) C33 porphyrins were quite

different from each other. The Ni(ll) complex showed [M-43]* as the most intense

of these ions (Figure 2-5). On the ternary diagram it lay in the domain of the

CAP-7 porphyrins (Figure 2-4) In contrast, the VO(ll) complex lay in the domain

of the CAP-5 porphyrins (Figure 2-4).

The daughter ion spectrum of the Ni(ll) C34 complex is less easily

interpreted (Figure 2-5, Table 2-2). The spectrum shows an intense [M-43]+

daughter ion, and the compound lay within the domain of the THB porphyrins in

the ternary plot (Figure 2-4) These data could conceivably indicate that the

porphyrin is a CAP-7 or THB type species; however, such speculation must be

treated with caution. The [M-43]' might also be the product ot f-cleavage of a

butyl or isobutyl moiety, which would sharply skew the fragmentation pattern.

Nevertheless, it is intriguing to note that in the daughter ion spectra of the

molecular ions of all higher carbon number (> C,) porphyrins studied from both

New Albany Shale and Boscan oil the [M-44]+ and [M-45] daughter ions were

always of very low relative abundance (Johnson et al., 1986; Quirke et al., 1989].

The above data demonstrate the potential value of tandem mass

spectrometry in the characterization of CAP geoporphyrins. It is essential to

perform chromatographic separations to avoid the problem of interpreting the

daughter ion spectra of unresolved isomers. The use of the ternary diagrams

may provide the researcher with a means of determining whether such a problem

has arisen. Clearly, on-line high performance liquid chromatography/tandem

mass spectrometer (LC/MS/MS) would be an ideal way to effect the analysis of

geoporphyrin mixtures.



The EIMS/MS daughter ion spectra of the molecular ions of CAP porphyrins

have been obtained. The [M-43]+, [M-44]* and [M-45] daughter ions are the

most valuable ions for distinguishing the skeletal type of CAP porphyrins. An

intense [M-44] + daughter ion relative to [M-43]' or [M-45]+ is indicative of a CAP-5

or CAP-5-Me porphyrin. CAP-6 porphyrins display [M-45]* as the most intense

of these daughter ions. CAP-6-Me porphyrins show [M-44]+ and [M-45]+

daughter ions in similar relative abundances. All three ions are present in the

daughter ion spectra of CAP-7 porphyrins with the [M-43]+ ion being the most

intense. The THB porphyrins are characterized by an intense [M-43]+ daughter

ion, the other two ions being present in very low relative abundance. The need

to separate the isomeric CAP porphyrins to avoid problems in interpretation of the

daughter ion spectra of the M' ions would make LC/MS/MS the ideal method for

the analysis of geoporphyrin mixtures.

In normalizing the intensities of the [M-43]+, [M-44]+, and [M-45]+ daughter

ions and plotting them in a ternary diagram, distinct clusters of data points

correspond to the various CAP skeletal types. When applied to data already

obtained for partially-separated mixtures of geoporphyrins, the technique showed

promise for the analysis of geoporphyrins. Most of the geoporphyrin data plotted

in the CAP-5/CAP-5-Me region of the ternary diagram. However, two of the data

points for high-carbon number porphyrins (the Ni(ll) C,3 and the Ni(ll) C,) plotted

in the CAP-7 and THB regions, respectively, of the ternary diagram. This

indicates CAP-7 and THB porphyrins may be present in Shales and oil deposits

(in opposition to the Treibs hypothesis); previous studies in our group [Johnson

et al., 1986] were unable to confidently propose the stuctures of the high-carbon

number porphyrins since standard compounds were not available at the time.



Porphyrins are of considerable importance in many areas of chemistry and

biology. For example, heme, the prosthetic group in hemoglobin and most

cytochromes, plays a crucial role in biological transportation of oxygen and

electrons respectively [Marks, 1969; Lemberg and Barret, 1973]. Perhaps the

most important property of porphyrin macrocycles is their ability to chelate with

virtually all the metallic elements [Buchler, 1975].

Spectroscopic Studies of Nitroporphvrins

It has long been known that the nature of the chelated metal ion, the

peripheral substituents, and the axial ligand can potentially alter the

spectroscopic, redox, and conformational properties of porphyrins. These

properties are of foremost importance in determining the activity of the porphyrins

in biological reaction centers. In particular, much research has been performed

recently to determine the importance of the non-planar conformation of many

biologically active porphyrins [Trunrud et al., 1986; Horning et al., 1986; Barkigia

et al., 1988; Deisenhofer, 1989]. An understanding of the structure-activity

relationship of distorted biological porphyrins aids in the development of

biomimetic photocatalysts [Shelnutt and Trudell, 1989].

Recent studies on meso-nitro-octaalkyl porphyrins have indicated that the

compounds are of considerable value as biological models. Medforth et al.

[1990; 1992] characterized sterically strained non-planar porphyrins containing

bulky peripheral substituents. However, the non-planarity of meso-nitroporphyrins

has brought them interest in the investigation of properties of non-planar

porphyrins. This is especially true since the synthesis of nitroporphyrins is

considerably easier than that for other non-planar systems and electron

withdrawing groups can facilitate axial ligation [Senge, 1993]. Anderson et al.

[1993] proposed that the small perturbation of the mono-nitro porphyrins may

more accurately represent the distortion present in the protein-bound environment

than the more highly substituted porphyrins. One prevalent example is the study

of cytochrome c [Shelnutt et al., 1992; Anderson et al., 1993; Hobbs et al., 1994].

Extensive spectroscopic studies have been performed on 5-nitro

octaethylporphyrin (5-NO2 OEP) and its divalent metal complexes [Gong and

Dolphin, 1984; Stanley, 1990;Wu et a/.,1991; Liu, 1993]. The effect of the

coordinated metal on the porphyrin macrocycle has been studied by various

spectroscopic techniques. Infrared (IR) [Ogoshi and Yoshida, 1971; Ogoshi etal.,

1971; Stanley et al., 1993], ultraviolet-visible (UV-visible) [Nappa and Valentine,

1978; Wang and Hoffman, 1984], and resonance Raman (RR) [Shelnutt et al.,

1991a,b] spectroscopies have received considerable attention in this regard. In

the latter part of this chapter, the effect of the chelated metal on the daughter

spectra of the molecular ions of metallated NO,-OEPs will be described and

compared to the results obtained through other spectroscopic techniques.

Mass Spectrometry of Nitroporphvrins

Porphyrins have been of interest in mass spectrometry as they have proved

useful in investigating a variety of phenomena. The high relative abundance of

doubly charged ions occurring in electron ionization mass spectrometry (EIMS)

of porphyrins and their fragmentation pathways were studied by Beato et al.

[1989a]. In chemical ionization mass spectrometry (CIMS), surface-induced

decomposition of porphyrins into mono-, di-, and tripyrrolic units has been

observed [Shaw et al., 1981; Beato et al., 1989b; Van Berkel et al., 1989a,b].

Recently, Van Berkel et al. [1992] reported that Ni(ll) porphyrins undergo

electrochemistry in the electrospray needle in electrospray ionization mass

spectrometry (ESMS). Yan et al. have studied the effect of the meso substituent

on the linear dependence of the appearance of [M+H]* ions (basicity) in fast-

atom bombardment (FAB) mass spectrometry of OEPs [1991]. The effect of

metal ions on ammonia Cl mass spectra of metalloporphyrins has been

investigated [Beato et al., 1989b] In all of the above studies, the characteristics

of the chelated metal ion, if any, modified the mass spectra.

There have been only limited reports on the EIMS analysis of meso (bridge)

substituted porphyrins. Jackson et al. presented the first detailed paper on the

mass spectrometry of porphyrins in 1965. Budzikiewicz [1978] performed a few

studies on the mass spectrometry of meso-substituted porphyrins. In neral it

was believed that the presence of a meso substituent did not modify the

fragmentation pathway significantly. However, the studies presented in Chapter

2 of this dissertation on cyclo-alkano-porphyrins (CAP) indicate that unusual

fragmentations can occur. Clezy et al. determined that the presence of a nitro

group at one of the bridge carbons modified the EIMS spectrum substantially

because the normally encountered f-cleavage of pyrrolic alkyl substituents was

suppressed [1974]. The fragmentations may be a result of the meso substituent

or of the conformation of the isocyclic ring as a whole [Jackson et al., 1965; Clezy

et al., 1974; Smith, 1975; Budzikiewicz 1978]. Investigation of such novel

fragmentations by electron ionization tandem mass spectrometry (EIMS/MS) is of

fundamental interest since it may be possible to relate the mass spectrometric

data to other characteristic spectroscopic properties.

The mass spectra of metalloporphyrins are markedly influenced by the

nature of the chelated metal ion. Beato et al. [1989b] showed that the relative

abundance of doubly charged ions to singly-charged ions in the El mass spectra

of metallated complexes of octaethylporphyrin OEP is related to the chelated

metal (as is discussed in Chapter 1 of this thesis). The singly charged region of

the mass spectrum, however, remains relatively unaffected by the identity of the

chelated metal ion. Undoubtedly, the role of the metal ion in the mass spectra

of metalloporphyrins warrants extensive investigation.

The present study is limited to an investigation of the divalent metal

complexes of porphyrins for two reasons. First, trivalent or tetravalent complexes

bear axial ligands that are often exchangeable. Thus, the homogeneity of such

complexes may be hard to guarantee. Second, it is known that higher valent

metal complexes may be transformed into divalent metal complexes during

volatilization [Edwards et al., 1970].

In this Chapter are presented the results of EIMS, EIMS/MS and electron

ionization high resolution mass spectrometric (EIHRMS) analysis of 5-NO2

octaethylporphyrin. A novel fragmentation involving the scission of the porphyrin

macrocycle is presented and the implications are discussed. In contrast to the

mass spectra for OEP and its metal complexes, the insertion of metals into 5-NO,-

OEP was found to have a strong influence on the types of fragmentation

encountered in the singly charged region of the mass spectrum. The divalent

metal complexes studied include Co, Cu, Zn, Ni, Pd, Ag and Mg. The effect of

the metals on the fragmentations is to be discussed in detail; the relation of the

mass spectral data to other metal-dependent spectroscopic data is also




Compound Preparation

Synthesis of NO, porphyrins. 5-NO, octaethylporphyrin was prepared by

the method of Bonnett and Stephenson [1965]. The method involves the

electrophilic substitution of the nitro group at one of the bridge carbons using

fuming nitric and acetic acid as the nitrating agent. A mixture of 5-NO, OEP and

the 5,10 and the 5,15 di-NO2 isomers of OEP were obtained.

The 5-NO, was purified by column chromatography. Using the slurry

method, a ten inch bed of silica (200-400 mesh) was packed in 4:1

hexane/toluene. The compounds were then eluted with 4:1 hexane/toluene,

increasing the polarity as needed. The first fraction, which eluted with 3:1

hexane/toluene, contained the di-NO2 OEP isomers. As the polarity increased, the

5-NO2 OEP eluted in 2:1 hexane/toluene in a distinct band. The remaining

porphyrin band, which eluted with 1:1 hexane/toluene, contained the unreacted

OEP. The column was finally washed with dichloromethane and methanol. The

second fraction, which contained the pure 5-NO2 OEP, was evaporated under

vacuum and recrystallized from 1:1 dichloromethane/methanol.

Synthesis of isotopicallv labelled porphvrins. Octaethylporphyrin labelled

with '5N at all four porphyrinic nitrogens was prepared by the method of Callot et

al. using '1N ammonium chloride (Cambridge Isotopic Laboratories) as the initial

source of nitrogen [1983]. Octaethylporphyrin labelled with 13C at each of the four

bridge positions was also prepared by the method of Callot [1983]. Each of these

two isotopically labelled compounds were nitrated by the method of Bonnett and

Stephenson [1965] and purified and recrystallized as above.

The structure of 5-NO2 OEP (or meso nitro OEP) is shown in Figure 3-1.

The sites of isotopic labelling with 1C ar-" 'N have been marked with symbols

for clarification.

Synthesis of Ni(ll) and Co(ll) 5-NO, OEP. The 5-NO, OEP (50 mg) and

nickel (II) acetate (100 mg) were dissolved in dimethyl formamide (15 mL). The

solution was refluxed and was monitored by UV-visible spectrophotometry. When

the reaction was completed (after about one hour), the solution was diluted with

water and extracted with dichloromethane. The organic layer was separated and

washed with water. The organic layer was then evaporated under vacuum and

the porphyrin was recrystallized as described above. TLC analysis for Ni(ll) 5-NO2

OEP were carried out using hexane/toluene (1:1 ;v:v) as the mobile phase.

Co(ll) 5-NO, OEP was prepared by the same method using cobalt (II)

acetate. The solvent system had to be modified to dissolve the porphyrin and salt

by using 2 mL of dichloromethane and 10 mL of methanol. TLC was carried out

using hexane/toluene (1:1;v:v) as the mobile phase.

Synthesis of Zn(ll) and Cu(ll) 5-NO OEP. The 5-NO OEP (50 mg) was

dissolved in dichloromethane (25 ml) and a solution of zinc acetate (100 mg ) in

methanol was added. The mixture was refluxed for one hour and the product was

Et t


Et/- N Et
0 0

Et Et

Figure 3-1: Structure of 5-N02 OEP. Sites of 13C labelling indicated by ( o).
Sites of 15N labelling indicated by (*).

isolated by distilling off the dichloromethane and adding methanol. The Zn(ll) 5-

NO2 OEP was filtered off and recrystallized as described above. The purity of the

Zn(ll) 5-NO2 OEP was determined by TLC with hexane/toluene (1:1;v:v) as the

mobile phase.

Cu(ll) 5-NO2 OEP was synthesized as above using copper (II) acetate. TLC

analyses were carried out using hexane/toluene (1:1; v:v).

Synthesis of Mg 5-NO, OEP. The 5-NO2 OEP (50 mg) and magnesium

perchlorate (200 mg) were dissolved in pyridine. The mixture was refluxed for two

hours and the reaction was monitored by UV-visible absorption until complete.

Upon completion the pyridine was removed under vacuum and the product was

washed several times with water. The product was then dissolved in

dichloromethane which had been eluted through a grade V alumina column to

remove any traces of acid. The Mg(ll) 5-NO2 OEP was then recrystallized in

dichloromethane/methanol (1:1 ;v:v) which had also been filtered through alumina

to remove acid. TLC analysis for Mg(ll) 5-NO2 OEP was carried out using 10%

ethyl acetate in dichloromethane as the mobile phase.

Synthesis of Aq(ll) 5-NO, OEP. The mixture of 5-NO2 OEP (30 mg) and

AgNO3 (100 mg) was dissolved in DMF (7 mL). The solution was refluxed under

an argon atmosphere and monitored by UV-visible spectroscopy. After the

reaction was completed (ca. 15 min), the mixture was diluted with 100mL of water

and extracted with methylene chloride. The porphyrin went into the organic phase.

Any solid residue (primarily inorganic byproducts) was filtered off. The solvent

was evaporated and the residue was recrystallized with

dichloromethane/methanol (1:1;v:v).

Synthesis of Pd(ll) OEP. 5-NO, OEP (50 mg) and PdCI, (50 mg) were

dissolved in benzonitrile (7 mL), and the mixture was refluxed under argon

atmosphere until metallation was completed as determined by UV-visible spectra

(ca. 15 min). The mixture was chilled and vacuum filtered. The soli.: product was

recrystallized from pure toluene.


All mass spectra presented here were obtained with a Finnigan MAT TSQ

45 triple quadrupole mass spectrometer. Porphyrin samples were dissolved in

dichloromethane and 1 pL aliquots of the solution were deposited onto a direct

exposure probe (DEP) filament. The DEP was heated from 100 to 600 C at 600

C min1. The rapid rate of heating was found to reduce the amount of thermal

elimination of NO, observed in EIMS.

The spectra were obtained under standard El conditions. The filament

emission current was maintained at 0.3 mA and the electron energy was 70 eV

for all experiments. For the initial EIMS experiments quadrupole 1 (Q1) was

scanned from m/z 50 to 800 in 0.8 s with Q2 and Q3 passing all masses. Tuning

for MS/MS was achieved with Co(ll) OEP in a capped aluminum solids probe vial

heated to 2750C. The MS/MS experiments were performed with a collision gas

pressure of 1.6 mTorr (argon) and a collision energy of 24.8 eV.

A Finnigan MAT 95 high resolution mass spectrometer was used for

determination of exact masses for El fragment ions. The MAT 95 was tuned for

a resolution of 7000. In this case, the samples were desorbed from a water-

cooled solids probe. The high resolution data were used as an aid in determining

the composition of certain fragment ions.

Results and Discussion

The singly charged fragment ions of OEP arise primarily from a sequence

of successive /-cleavages of the peripheral ethyl groups (Figure 3-2a). This

fragmentation pathway was confirmed by analysis of the metastable ions by Clezy

et al. [1974]. The porphyrin macrocycle remains intact; doubly charged ions are

present in high relative abundance as would be expected. The work presented

here concentrates only on the singly charged region of the mass spectrum of 5-


Analysis of Free-base 5-NO, OEP

With a nitro group attached to the bridge carbon position, the

fragmentation becomes significantly altered. In our MS and MS/MS studies of

Figure 3-2:

El mass spectra of 5-NO2 OEP: a) MS of OEP; b) MS of 5-NO2
OEP; c) Daughter spectrum of the molecular ion of 5-NO OEP; d)
Parent spectrum of the m/z 375 ion of 5-NO2 OEP.


^ ,


free-base 5-NO2-OEP, both normal El and daughter ion spectra (Figures 3-2b and

3-2c) showed a significant [M-17]+ peak owing to loss of OH' as was reported by

Clezy et al. [1974] in their EIMS work. The mechanism that has been proposed

by Clezy et al. for the loss of OH' is shown in Figure 3-3. The elimination of a

hydrogen from the P-substituent adjacent to the nitro group occurs with a seven-
membered cyclic rearrangement, giving an ion at m/z 562.

From the El mass spectrum of 5-NO,-OEP (Figure 3-2b) we see that peaks

corresponding to /-cleavage ions (e.g. m/z 564) are minor, whereas loss of OH

(m/z 562) as well as a-cleavage (m/z 550) result in major peaks. A peak at m/z

533 has been attributed to loss of NO2.

We have found evidence for a novel ring fragmentation of 5-NO2-OEP. Our

EIMS analysis showed a significant peak at m/z 375 which we have attributed to

loss of a pyrrole unit. Such fragmentation processes for substituted porphyrins

have previously been observed only as a surface-induced fragmentation process

in CIMS [Beato et al., 1989a] (refer to Chapter 1 of this dissertation for the

detailed discussion of this phenomenon).

The EIMS analysis of 5-NO2 OEP is not easy to perform because the nitro

group may be thermally eliminated in the solids probe vial or ion source during

the heating required for volatilization, with intermolecular hydrogen abstraction

forming OEP. This problem can be avoided if it is possible to heat the sample

rapidly. Therefore the use of a direct exposure probe is to be recommended as

opposed to use of the solids probe. The ElMS of 5-NO2 OEP is shown in Figure



3-2b. The spectrum is broadly similar to that reported by Clezy et al. in 1974. In

the present study, there was little contamination from thermal decomposition of

the 5-NO, OEP, as evidenced by the very low abundance of the M'' for OEP (m/z


The singly charged region of the molecule is complex, and is substantially

different from that of OEP. The presence of the m/z 564 ion as a minor

component in both the ElMS and the EIMS/MS daughter ion spectrum of the M+'

ion of 5-NO2 OEP confirmed that the #-cleavage fragmentation pathway is

suppressed in this molecule. The eight most abundant fragment ions observed

were m/z 562, 550, 533, 523, 522, 504, 489, and 375. It was not obvious which

of these ions were produced directly from the molecular ion, M+' (m/z 579), or

from thermal decomposition occurring in the sample vial or in the ion source.

Therefore the ElMS/MS daughter ion spectrum of the M+" ion of 5-NO2 OEP was

obtained. The spectrum is shown in Figure 3-2c; the most abundant daughter

ions were m/z 564, 562, 550, 535, 533, 523, 522, 521, and 375. The origin of

each of these ions will be discussed in turn.

The [M-17]+ daughter ion, m/z 562, is formed by loss of a hydroxy radical

via a rearrangement such as that proposed by Clezy et al. [1974] and is

discussed above (Figures 3-2c and 3-3). The ions m/z 550, [M-29]+, and 533,

[M-46]+, are generated by a-cleavage of ethyl and nitro groups, respectively, and

are analogous to the same ions produced in the ElMS spectrum.

The origins of the daughter ions m/z 535, 523, 522, 521 and 375 are more

Difficult to assign. In fact, it was essential to obtain electron ionization high

resolution mass spectra (EIHRMS) to determine the elemental composition of the

neutrals lost and the daughter ions. Table 3-1 shows the exact mass, A (the
difference in millimass units (mmu) from the actual mass of the ion reported),

percent relative abundance, and the ion composition for the ions of interest in the

mass spectra of 5-NO2 OEP and its isotopically labelled analogs. For the 3C4 and

'5N4 labelled compounds, only the ions pertinent to the ring cleavage process are

shown. The EIHRMS analyses confirmed the interpretation of the m/z 562, 550

and 533 daughter ions. Unfortunately, although the m/z 535, (M-44)+ ion is quite

abundant in the EIMS/MS spectrum (Figure 3-2b), it is of very low relative

abundance in both the EIMS and the EIHRMS spectrum; therefore, it was not

possible to determine either the elemental composition or the pathway to this ion.

The m/z 522 daughter ion is the result of a loss of C3HsO from the M' ion. To

account for this ion, it is necessary to invoke a cleavage of a carbon from the

macrocycle, as is shown in Figure 3-4. The mechanism of this fragmentation will

be discussed inmore detail below, as it appears that the m/z 522 ion is an

important intermediate in the ring scission process (see Figure 3-5) and the

formation of the m/z 375 ion. A similar pathway may be used to explain the

formation of the ion m/z 521 (M-58), in which the neutral lost has the elemental

composition C3HO. The m/z 523 ion is the 13C peak for the ion at m/z 522. The

relative intensities of the two ions confirm this. The most intriguing daughter ion

a s

0) 0)


cSo :
I5 < i5
^ "S -
0 >0


c .2 0
o 0 0
0 a 0 0
E E e E

I .2
0 c

E 75






S (

g 0

_ z qo o o o z

0 u 0 0

t 0- d
'~t~ t U C' 1 ~ -

03))10~ d1 OC'J N

30 n p
10 q

to i U)
in Si i

0 0 CM 10 CM
oc' cJ CM

11 o 0 10 mo

C) U)M ) o 10
a mno in l n

ob b

y, C



0 0) Cl
0 -: 0
0 10 0

o Lo 0

10 CM1 N
11 i0 C

i 0 0 C





- m

U i
0 0
I o










._ cv
T" <

it~j c
L~Ej c3

is m/z 375. This ion has the elemental composition C24H29N30, i.e. it is formed by

loss of C1,,HN0O. The loss of two nitrogens implies that the ion must be formed

by cleavage of at least one pyrrolic ring. Such fragmentation processes have

previously been observed for substituted porphyrins only as a surface-induced

fragmentation process in CIMS [Beato et al., 1989a].

In order to obtain more informa on on the pathway to the formation of the

m/z 375 ion, the MS/MS parent ion spectrum of was obtained (Figure 3-2d). This

indicated that the molecular ion was the major parent ion and that the ions m/z

522 404 and 390 were also parents. The m/z 404 parent supports the idea that

an ethyl group from another pyrrolic ring is a part of the fragment that is lost in

the formation of m/z 375. The absence of m/z 562, m/z 533, and m/z 550 in the

parent spectrum indicates that molecular ions losing OH', NO,, or C2115 will not

fragment directly to give m/z 375 to any significant extent.

The analyses were repeated using 13C and '5N isotopically labelled

porphyrins. In order to confirm that only one pyrrolic nitrogen is lost to form the

m/z 375 ion, 15N, labelled 5-NO, OEP (Figure 3-1) was run by HRMS. A fragment

ion appeared at m/z 378.2210 indicating a composition of C24H29015N3; the loss

of C1,H,,ON'5N indicates that the nitrogen of the nitro group and the nitrogen of

one of the pyrrole groups is lost. The data for the "C4 labelled compound

(Figure 3-1) confirm this interpretation. The HRMS spectrum shows an ion at

378.2400 with the composition C2, H2013CAN, (Table 3-1). The fragment lost has

the composition C,,H,,13CON,; the obvious routes to this loss involve the cleavage

of one pyrrolic unit, one bridge carbon and the nitrogen of the nitro group.

A proposed scheme for the formation of the m/z 375 ion in the EIMS

spectra is shown in Figure 3-5; double arrows indicate rearangement steps. The

HRMS accurate mass assignments indicate that the ions at m/z 522 and m/z 375

occur from losses of C03H50 and C,,H,,N20, respectively (Table 3-1) The m/z 522

ion is the major ion in the parent of the ion at m/z 375. The data for the 13C4

labelled 5NO OEP indicate that the meso-carbon is not lost in the pathway (Table

3-1). Therefore, one of the carbons lost is likely a #-carbon from the pyrrolic ring

adjacent to the nitro group. If the #-carbon is lost, then it is most likely that the

nitro group will transfer an oxygen to the #-carbon of the porphyrin, activating the

ring towards elimination. It is unlikely that abstraction of a hydrogen atom from

the alkyl group by an oxygen of the nitro group is unlikely to be an initial stage

in the pathway because m/z 562 does not appear as a parent ion of of m/z 375.

Other unlikely possibilities that have not been eliminated by MS/MS studies are

the loss of CH3, together with HCHO or the loss of C2H5' together with CO. As

is shown in Figure 3-5, one possibility is that a pyrrolic ring might cleave forming

a ketone, followed by loss of C3H50' via homolytic cleavage. Rearrangement of

the m/z 522 ion can lead to the aromatic ion shown in Figure 3-5. This could

eliminate in stepwise fashion with loss of ethyl or methyl forming intermediate ions

at m/z 390 and 404 respectively, followed by the expulsion of the remainder of the

pyrrolic unit. Alternatively, elimination may also occur in one step with loss of the

cyanopyrrole radical to generate m/z 375 ion.


v-,. +

+d I

z z

I U)





0'' 3 1
-" E
,N U) a

+ v*


There are three possible explanations for the differences in the

fragmentation pathways between OEP and 5-NO, OEP. First, the nitro group may

be more labile than the alkyl groups, hence the #-cleavage of the alkyl groups is
suppressed because the nitro group fragments more readily than the alkyl

groups. This explains the predominance of [M-17] and [M-46]+ ions in the

spectrum. This does not, however explain the a-cleavage of the alkyl moieties or
the cleavage of the macrocycle. If the NO, does not modify the properties of the

macrocycle this does not explain the appearance of the m/z 375 ion. A second

possibility is that the NO, group causes the macrocycle to become non-planar,

thereby significantly changing the fragmentation process.

Structures of nitro-porphyrins obtained with X-ray crystallography clearly

show the nonplanarity of the nitro porphyrins [Wu et al., 1991; Shelnutt et a.,

1992]. In their characterization of 5-NO2 OEP using 'H NMR, Bonnett and

Stephenson [1965] showed that the structure is non-planar in solution. The

conformation of protonated 5-NO2 OEP has been shown to be especially distorted

owing to both the electrostatic repulsion of the positively charged pyrrolic

nitrogens and the steric interactions between the nitro group and the adjacent f-
subtituents [Meot-Ner and Adler, 1975]. Gong and Dolphin [1984] showed similar

results in their UV-visible spectrophotometric characterization of nitroporphyrins;

the addition of a nitro group to OEP causes a bathochromic shift and a

weakening of all of the visible absorption bands. Theoretical calculations show

that a direct correlation exists for the degree of macrocycle distortion and the

bathochromic shifts exhibited in the absorption spectrum of porphyrins [Fajer et

al., 1985]. Using the principles of Goutermans "four orbital" theory [1978] and UV-

vis absorption and emmission data, Wu et al. studied porphyrins bearing a meso

sustituent [1991]. They showed that apart from the electronic effects of the

substituent itself (the presence of electron-donating groups raises the energy of

the highest occupied molecular orbital (HOMO) while electron-withdrawing groups

lower the energy of the HOMO), the steric effects were substantial in red-shifting

the absorption bands.

We propose that the gas-phase molecular ion of 5-NO2 OEP retains its

distorted shape and that the positive charge may even accentuate this effect. The

non-planarity disrupts the conjugated t-bonding system of the macrocycle
sufficiently to produce a significant weakening of the bonding system of the

macrocycle. This could account for the cleavage of the pyrrolic ring leading to

the observance of the m/z 375 ion in the spectrum. It is well known that cyclic

tetrapyrroles which are not fully aromatized readily undergo cleavages of pyrrolic

units [Boylan, 1969; Budzikiewicz, 1978]. Other evidence is that 5-NO2-OEP

shows anomalous chromatographic behavior in that it is much less polar than

OEP in normal phase chromatography (indicating a disruption of the n-system)
[Bonnet and Stephenson, 1965].

Analysis of the Divalent Metal Complexes of 5-NO, OEP

Spectroscopic studies

There are many reports of systematic studies on the effect of the chelated

metal ion and its axial ligands on the spectroscopic properties of porphyrins [Falk,

1964; Gouterman, 1978]. Nappa and Valentine [1978] studied the effect of axial

ligands on the visible absorption spectra of zinc porphyrins. Wang and Hoffman

[1984] studied the trends in the optical spectra of several biologically important

metalloporphyrin enzymes. Spaulding et al. [1975] were the first to show the

dependence of certain resonance Raman frequencies on the core size of

metalloporphyrins. Parthasarathi et al. [1987] studied trends in the resonance

Raman (RR) frequencies of several metalloporphyrin derivatives with an emphasis

on the implications for the interpretation of hemoglobin photoproduct RR


Stanley et al. [1993] have studied the metal-dependent bands in the IR

spectra of a series of divalent metal complexes of OEP as dispersions in cesium

iodide pellets. By studying the porphyrins in the solid state, it was possible to use

X-ray crystallographic data with more confidence. As a result of this study, it was

possible to draw a meaningful correlation between metal-pyrrolic nitrogen bond

distances (from X-ray) and precise peak positions of metal dependent bands in

IR. The Ceso-H bond vibrations (e.g., IR bands ca. 3050 and 1230 cm1) were the

most conformationally sensitive, as evidenced by the distinctly different IR

absorbances for the planar and ruffled (non-planar) forms of OEP.

Anderson et al. [1993] compared the RR frequencies of a series of metal

derivatives of OEP and of 5-NO2 OEP. They observed that the metal porphyrin

complexes including the Ni(ll) undergo changes in conformation in solution.

Furthermore it was possible to relate both the planar-nonplanar equilibrium and

the position of Raman structure sensitive marker bands to core size. The most

sensitive marker band, vi0, involves the C,-CmeO-Ca bond vibrations. These

relationships stem from the fact that the porphyrin must contract or expand in

order to chelate with metals either smaller or larger than the optimum size. The

study also indicated that the nitro stretching vibrations are coupled to vibrational

modes for the porphyrin macrocycle.

Molecular modeling as well as X-ray crystallographic data were used to

determine the core sizes for the complexes [Anderson et al., 1993]. The

modelling data also indicated that the repulsions between the nitro group and the

adjacent ethyl groups contribute to an increase in the core size. It was reported

that the core size is an indicator of planarity; thus the RR frequency shifts can be

said to be measure a of the planarity of the macrocycle [Anderson et al., 1993].

Mass spectrometric studies

In the EIMS analysis of octaethylporphyrin (OEP) and its metal complexes,

the metal was found to be important in determining the relative abundance of ions

in the singly charged region and doubly charged region of the mass spectrum

[Beato et al., 1989b]. In contrast, the fragmentation patterns in these regions

were not substantially influenced by the nature of the metal ion. The ratio

between the summation of doubly-charged fragment ions and the summation of

singly charged fragment ions (IF2+/F+) and a modified Buchler stability index (Si,

see chapter 1) for the inserted metal ion was plotted, and the results indicated

that there might be some correlation between these parameters [Beato et al.,

1989b]. The fragmentation patterns observed were essentially otherwise

unaffected by the metal ion; the abundance of singly-charged #-cleavage
fragment ions, EF+, is relatively unaffected by the metal when compared to the

changes in the abundance of doubly charged fragment ions, F2'.

In the EIMS spectra for the divalent metal complexes of 5-NO2 OEP, the

effect of the metal on the ratio EF2+/iF+ was not pronounced. The ratio showed

no clear correlation when plotted versus Si. The intensities of certain singly

charged El fragment ions appeared to have a significant dependence on the

identity of the inserted metal ion. Also, no ions corresponding to the loss of a

pyrrole unit are observed in EIMS for any of the metallated 5-NO2 OEPs studied.

In order to more closely study the metal dependence on the singly charged

fragment ions of the divalent metallated 5-NO, OEPs, MS/MS and HRMS data

were obtained. The daughter ion spectra for the metalloporphyrins were

examined to determine how the metal alters the fragmentation of 5-NO, OEP. The

systematic trends in the fragmentations were related to inherent physico-chemical

properties of the inserted metal and to spectroscopic data in order to determine

if a correlation exists.

The El MS/MS fragmentation observed for 5-NO,-OEP was significantly

altered by insertion of a divalent metal. The silver complex of 5-NO2 OEP behaved

anomalously and will be discussed separately. The daughter spectra for the

molecular ions of the Mg(ll), Cu(ll), and Co(ll) are shown in Figure 3-6 a,b, and

c, respectively. Ions at [M-15]+ and [M-17] corresponding to the loss of CH3'

and of OH' were detected for all of the metals, as for metal-free 5-NO2 OEP. In

contrast to the metal-free 5-NO, OEP, there was no evidence of pyrrole ring

fragmentation for any of the complexes studied. Significant ions at [M-75]+ and

[M-86] were noted for all of the metals. Both of the corresponding ions were

minor in the daughter spectrum of metal-free 5-NO2 OEP.

In view of the complexity of the fragmentation pathway for 5-NO, OEP, it

was necessary to confirm the interpretation of the fragmentation pathways for the

metalloporphyrins by HRMS. Thus, Co(ll) 5-NO2 OEP was analyzed by this

technique. Unsurprisingly, the [M-15]+ and [M-17] were confirmed as being due

to losses of CH,' and OH', respectively. The [M-75] ion had a composition of

Figure 3-6: Selected EIMS/MS daughter ion spectra of the M+" ions of
metallated complexes of5-NO2 OEP: a) Mg(ll) complex (m/z 601);
b) Cu(ll) complex (m/z 640) c) Co(ll) complex (m/z 636); d) Ag(ll)
complex (m/z 684).




>eP e (8







C34H38N4Co (exact mass 561.2381, A 4.7 mmu), which confirmed our interpretation

of the loss of C2H5' and NO2. The [M-86] ion had a composition of CH,33ONsCo

(exact mass 550.2013, A 0.4 mmu), which corresponds to a loss of CsH,00. The

fragmentations leading to the [M-86]+ ion are interpreted in more detail below.

The [M-75]' ion resulted from loss of NO2 and C2H5' (Figure 3-7). Note

that the molecular ion is not iricated as a radical since this is dependent on the

identity of the metal. The daughter spectra of [M-75]+ ions had abundant peaks

corresponding to /3-cleavage. The relative abundance of the collision-induced
dissociation (CID) fragment ions of the [M-75]+ ions were relatively independent

of the inserted metal. This is to be expected because once the nitro group is lost

the compound should behave similarly to OEP. This supports the HRMS data for

the composition of [M-75]+. In the parent spectra of [M-75] ions, the molecular

ion was the only detected ion. This suggests that the loss of NO2 and C2,H" are

concerted within the time scale of CID, as shown in Figure 3-7.

The [M-86]+ ions resulted from the neutral loss of C5HliO. This provides

strong evidence that migration of at least one oxygen is an important step in the

fragmentation. Interestingly, parent spectra of the [M-86]+ ions showed that

molecular ions were the only detectable parent ions. It is not clear how this

unusual fragment ion arises. It would seem likely that an oxygen of the nitro

group migrates and activates the cleavage of the porphyrin macrocycle. We have

tentatively proposed a fragmentation pathway that accounts for the elemental

composition of the daughter ion and also results in the generation of a new 22 n-
electron macrocycle (Figure 3-8), which could conceivably display mass




spectrometric behavior similar to OEP. The daughter spectra of the [M-86]1 ions

showed intense peaks corresponding to #-cleavage of peripheral ethyl groups.

It is possible that the chelated metal ion could be penta coordinate in the [M-86]'

ion. Clearly, the oxygen-activated cleavage of the porphyrin macrocycle is of

importance to both metal-free and metal complexes of 5-NO, OEP because the

[M-57]' ion (m/z 522) in the metal-free porphyrin is the product of the loss of a

C3H5O group, which probably is formed via a similar process.

For metal complexes, the loss of 204 to give a ring-cleavage product was

not observed. However, the loss of 86 may be the result of a similar pathway to

that for m/z 522 (Figure 3-8). The difference is that there is loss of C2H5'

(presumably lost via a-cleavage) in addition to the loss of C3HO'. The reason

there is no further fragmentation of the pyrrole ring is probably the templating

effect of the chelated metal ion.

One of the more unexpected features of this study is that the fragmentation

pathway of silver (II) 5-NO,-OEP is completely different from those of the other

complexes studied. The daughter ion spectrum, shown in Figure 3-6d,differs

significantly those of the other metals (see Figures 3-6 a,b, and c, for example).

The [M-75]' ion dominates the spectrum with the M+ ion being the only other ion

with a relative abundance greater than five percent. One possibility we have

considered to explain this behavior is that silver might assume a monovalent state

during the volatilization process; however, we did not detect any ions

corresponding to loss of silver which would likely result from such a process. It

should be noted that silver has the largest ionic radius of the metals studied,

which could be one factor that causes the anomalous behavior of the ion.

The [M-15], [M-17]+, [M-75]+, and [M-86]+ daughter ion relative

abundances (% RIC) have been plotted as a bar graph in order to more clearly

determine the metal dependency of each of these ions (Figure 3-9). The [M-17]+

ion abundance seemed to be relatively independent of the metal while the [M-

75]+ ion showed a small degree of metal-dependency. The [M-86] and the [M-

15]' ions showed the highest degree of dependence on the metal. The relative

abundance of the f-cleavage peak at [M-15]+ was influenced by the presence of

various metals, and was inversely related to the intensity of the [M-86]' peak

(Figure 3-9). Evidently, if a metal induces 3-cleavage, the fragmentation pathway

leading to [M-86] is inhibited.

The relative abundances of these daughter ions were plotted versus the

properties of the inserted metal ions to determine whether a significant

relationship exists. The properties plotted were the Pauling electronegativity, the

ionic radius, the number of d-electrons, and the Buchler stability index. The only

property that showed a relationship to any of the ion intensities was the number

of d-electrons, as discussed below. The intensities of the daughter ions were

plotted against several of the metal-dependent spectroscopic properties: IR band

shifts, UV-visible absorption band shifts (the Soret, a, and # bands), resonance
Raman marker band shifts, and X-ray structural information (core sizes). The IR

bands and the Raman lines showed a trend when plotted against the intensity of

the [M-86]' ion, as discussed below.