TANDEM MASS SPECTROMETRIC STUDIES OF PORPHYRINS:
STRUCTURAL AND PHOTOCHEMICAL STUDIES
JOHN D. LAYCOCK
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
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.
TABLE OF CONTENTS
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
2 CHARACTERIZATION OF CYCLOALKANOPORPHYRIN SKELETAL TYPES
USING ELECTRON IONIZATION TANDEM MASS SPECTROMETRY:
IMPLICATIONS FOR ANALYSIS OF GEOPORPHYRIN
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
3 ELECTRON IONIZATION TANDEM MASS SPECTROMETRY OF 5-NO2
OCTAETHYLPORPHYRIN (OEP): UNUSUAL FRAGMENTATIONS
AND THE INFLUENCE OF CHELATED DIVALENT METAL IONS ....
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 .......................... ..
4 TANDEM MASS SPECTROMETRIC STUDIES
SENSITIZING REACTIONS OF PORPHYRINS
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
TANDEM MASS SPECTROMETRIC STUDIES OF PORPHYRINS:
STRUCTURAL AND PHOTOCHEMICAL STUDIES
John D. Laycock
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
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 (
I i II
I I I a
:, 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
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
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;
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.
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:
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 C
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
z z z
35 C _; E -
zo z o
z z -= 0Z
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.  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
N E a) .
0 -r 4
I i i fi I i i
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
CHARACTERIZATION OF CYCLOALKANOPORPHYRIN SKELETAL TYPES USING
ELECTRON IONIZATION TANDEM MASS SPECTROMETRY: IMPUCATIONS FOR
ANALYSIS OF GEOPORPHYRIN MIXTURES
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
The geoporphyrins are a potentially valuable compound class for the
fingerprinting of oils in oil-oil and oil-source rock correlation studies in oil
DPEP = CAP-5
(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,
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 . 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.
Nickel porphyrins were prepared by treatment of the metal-free porphyrin
with excess Ni(ll) acetate in refluxing acetic acid as described by Buchler .
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.  or in
N,N-dimethylformamide as described by Adler et al. .
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
'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
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).
in the [M-43]* to [M-45] region of the spectrum varied less than 10% in
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
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
M-43 M-44 M-45
SThe ions were renormalized to [M-15]+ = 100% for comparison with the data in
** Re-normalized so that 1[M-43]+ + [M-44]+ + [M-45]+ = 100% for ternary plot
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.
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
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.
ELECTRON IONIZATION TANDEM MASS SPECTROMETRY OF 5-NO2
OCTAETHYLPORPHYRIN: UNUSUAL FRAGMENTATIONS AND THE INFLUENCE
OF CHELATED DIVALENT METAL IONS
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.
 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.  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 . 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  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 . 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
Synthesis of NO, porphyrins. 5-NO, octaethylporphyrin was prepared by
the method of Bonnett and Stephenson . 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 . Octaethylporphyrin labelled with 13C at each of the four
bridge positions was also prepared by the method of Callot . Each of these
two isotopically labelled compounds were nitrated by the method of Bonnett and
Stephenson  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
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/- N 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
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
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. . 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
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.  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.  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
I5 < i5
^ "S -
c .2 0
o 0 0
0 a 0 0
E E e E
_ 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
to i U)
in Si i
0 0 CM 10 CM
oc' cJ CM
11 o 0 10 mo
Cl Cl CO CMO
C) U)M ) o 10
a mno in l n
0 0) Cl
0 -: 0
0 10 0
CO CO Ci
o Lo 0
10 CM1 N
11 i0 C
i 0 0 C
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.
0'' 3 1
,N U) a
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  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  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  and UV-
vis absorption and emmission data, Wu et al. studied porphyrins bearing a meso
sustituent . 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
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  studied the effect of axial
ligands on the visible absorption spectra of zinc porphyrins. Wang and Hoffman
 studied the trends in the optical spectra of several biologically important
metalloporphyrin enzymes. Spaulding et al.  were the first to show the
dependence of certain resonance Raman frequencies on the core size of
metalloporphyrins. Parthasarathi et al.  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.  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.  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.