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the collision cell cID spectra than in the on-resonance cID
spectra, indicating again a time effect because of the low
internal energies of the ions formed in the ESI process. Both
collision cell and on-resonance cID spectra of 1a and 1b give
rise to signals from the steroid rings. In general, the losses of
c2H4 or c3H6 molecules after the elimination of the first water
molecule are observed. In the case of 1a, the loss of c4H8 or
(c2H4)2 can be identified with less abundance as well as the
[M+H–2H2o–2c2H4]+ ion.
2. Protonation at the hydroxyl groups at positions 17 and 16
(ring D) can occur. Investigations of samples with the same
structural motif, such as the 1,2-cyclopentadiol, yield a proton
affinity of 885kJmol−1. this rather high value is linked to a
possible synchronous stabilization of the proton by both oxygen
atoms at ring D. However, both hydroxyl groups of estriol’s
five-membered ring are situated in a trans relationship, so
this value should be significantly reduced, possibly almost to
the value of the freely rotating ethane diol with 816 kJmol−1
for comparison and better understanding of fragmenta- (Δ = −69kJmol−1). unfortunately, the proton affinity of a cyclic
tion within the steroid skeletal structure, the cID spectra of
estradiol and estriol have also been recorded. Estriol spectra
exhibit intense signals for the protonated molecular ion and
the loss of two water molecules. this clearly demonstrates
the need for an unsaturated substituent at the phenolic
hydroxyl group to induce the third water elimination. In the
case of estradiol both hydroxyl groups are eliminated as
water molecules. In this molecule, the hydroxyl groups are
attached to position 16 and 17 at the steroid ring D. this
proves the loss of the two water molecules must occur from
that ring in estradiol.
trans-pentane diol has not been published, but this value
should not be far away from the values of the stereoisomers of
1,3-cyclohexane diols which result in a difference of 54kJmol−1
(cis, 882kJmol−1; trans, 828kJmol−1) between the cis and trans
position of the hydroxyl groups.
3. comparing the proton affinities for the estradiols, the
tendency of attachment should be even more abased, since
the hydroxyl group at position 16 in the D ring is missing.
therefore the proton affinities should be comparable to
cyclopentanol, yielding a value around 795kJmol−1.
4. transferring the proton to the ether oxygen at ring A yields
a structure similar to the protonation of anisole or methoxy-
benzyl ether. Proton affinities for these compounds are given
to understand these differences in fragmentation reactions
in both sets of cID spectra, the question of the addition of
the proton in the ESI process must be addressed. In general, in the literature with values of 840kJmol−1 and 817kJmol−1.
protonation can take place at the oxygen atom(s) of the hydroxyl
group(s), the ether oxygen bridge or the unsaturated systems
in ring A or in the alkenylic moiety. the principal location of
the proton will be controlled by the local proton affinities of
the different structural parts of the steroids, as demonstrated
in Scheme 4. Although exact values are not available, data of
comparable structures from the work of Hunter and Lias14
have been used.
As a result of these comparisons, it is likely that protona-
tion in the estratriols will preferentially occur at the hydroxyl
groups at ring D and, to a lesser extent, at the ether attached
to ring A. In the case of different estradiols, a clear distinc-
tion between both positions is less evident. Since the proton
affinities for the hydroxyl at ring D and the ether group are
nearly similar, the protonation can take place at both sides,
possibly with some increased tendency to attach to the ether
bridge. An exchange of the attached proton to either side can
be accomplished by a long-range proton transfer through the
well-known Longevialle process15–17 found with bifunctional
steroids.
to summarize these results, the following conclusions can
be drawn.
1. the direct transfer of protons to the unsaturated system of
either the steroid ring A or the unsaturated side chain does
not seem to take place because the proton affinities of toluene,
benzene and propylene yield 778 kJ mol−1, 759 kJ mol−1 and
Scheme 5 summarizes the mechanistic considerations
leading to the loss of water molecules as well as the ether
751kJmol−1kJmol−1, respectively. this is lower than the affini- group from the different steroids investigated. Based on the
ties of all other possible positions.
suggestions above, the proton is initially attached to the hydroxyl
groups at the steroid ring D. the loss of two water molecules
upon protonation is a well-known reaction in 1,2 diols.18 Winkler
and McLafferty,19 as well as Hua et al.,20 could demonstrate that
1,2-cyclopentandiol loses two water molecules under chemical
ionization conditions leading to a protonated cyclopentadiene, in
our case to an allylic carbenium ion. furthermore, the authors
demonstrate that the loss of the second water molecule occurs
with higher abundances if both hydroxyl groups are in a trans
configuration which is also present in all investigated estratriols.
As indicated in Scheme 5, the loss of two water molecules leads
to the formation of double bonds and, therefore, to a protonated
cyclopentadiene moiety.
H+
OH
H+
H+
H
(OH)
H
H
O
In the case of the estradiols, protonation in the ESI process
does not only occur at the hydroxyl group at ring D but also at
the ether group. the relation of proton affinities for comparable
molecular structures suggests the protonation of the steroids
H+
Scheme 4. Possible protonation sites of the different steroids.
Freudenhammer, Christoph
