Photodegradation of oxytocin and thermal stability of photoproducts

Article abstract of DOI:10.1002/jps.23204

Photodegradation of oxytocin at λ = 253.7 nm and λ ≥ 290 nm results in the transformation of the intrachain disulfide bond into predominantly dithiohemiacetal and thioether. Especially the dithiohemiacetal is sensitive to further degradation by light and/or elevated temperature, implying that the combination of an initial photostress and a subsequent heat stress can yield products significantly different compared with those observed under heat stress only.

Full text of DOI:10.1002/jps.23204

RESEARCH ARTICLE
Photodegradation of Oxytocin and Thermal Stability
of Photoproducts
¨
OLIVIER MOZZICONACCI, CHRISTIAN SCHONEICH
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047
Received 15 February 2012; revised 27 April 2012; accepted 1 May 2012
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23204
ABSTRACT: Photodegradation of oxytocin at λ = 253.7 nm and λ ≥ 290 nm results in
the transformation of the intrachain disulfide bond into predominantly dithiohemiacetal and
thioether. Especially the dithiohemiacetal is sensitive to further degradation by light and/or
elevated temperature, implying that the combination of an initial photostress and a subse-
quent heat stress can yield products significantly different compared with those observed under
heat stress only. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
J Pharm Sci
Keywords: mass spectrometry; stability; oxidation; hormones; peptides
did not report any modifications of oxytocin under
daylight exposure. However, in the same report, it
is mentioned that oxytocin was protected from sun-
shine. UV wavelengths shorter than 290 nm are ab-
sorbed by molecular oxygen, ozone, and water vapor
in the upper atmosphere, and do not reach the sur-
face of the earth in measurable amounts. Thus, the
WHO has reported in their Environmental Health
Criteria15 that all biologically important effects of
sunlight are due to wavelengths λ > 290 nm. The spec-
tral irradiance of sunlight between 290 and 380 nm is
approximately between 0.5 and 1 W/(m2 nm).15 For
comparison, in our photochemical study, the irradi-
ance for λ > 290 nm is 60-fold to 100-fold higher than
natural sunshine.
Here, we provide a product analysis of oxytocin
exposed to UV light at λ = 253.7 nm and at λ ≥
290 nm. Photolysis at these wavelengths resulted in
the transformation of the intrachain disulfide bond
into a dithiohemiacetal structure, as expected based
on our earlier studies with several model disulfides,16
cyclic peptides,17 insulin,18 and IgG1.19 Such dithio-
hemiacetals are photolabile and heat labile.17 In fact,
prolonged UV exposure or incubation of the dithio-
hemiacetal to elevated temperatures led to the for-
mation of secondary products such as thioethers. All
products of oxytocin photodegradation have been ana-
lyzed by mass spectrometry (MS), including (MS–MS)
MS2 and MS–MS–MS (MS3) analysis. We discuss in
detail the fragmentation pathways of the cyclic frag-
ment ions obtained during MS2 and MS3 analysis. The
characterization of cyclic peptide structures through
INTRODUCTION
Peptide hormones are active in various physiological
processes and are critical for cell-signaling mecha-
nisms. Oxytocin is a neuropeptide used as a thera-
peutic drug for stimulating labor, lactation, and to
reduce the incidence of postpartum hemorrhage.1–3
More recently, a role for oxytocin in therapeutic in-
terventions in mental disorders associated with social
deficits has been discussed.4,5 The stability of oxytocin
is pH dependent with an optimal pH between 3 and
5.6,7 Degradation and specifically reduction–oxidation
studies of oxytocin have been performed to demon-
strate the cyclic structure of this neuropeptide.8,9 Sta-
bility studies of oxytocin have shown that any in-
jectable or solid formulation is rapidly degraded dur-
ing storage under conditions of elevated temperature
(>30◦C) and humidity.10,11 Several strategies have
been developed to stabilize oxytocin, for example, sta-
bilization by divalent metal ions.12 Although the ef-
fects of physical and chemical12,13 stresses on oxytocin
(e.g., pH and temperature) were extensively studied,
a detailed photodegradation study [under ultraviolet
(UV) light] of oxytocin has never been reported. In a
report on the stability of injectable oxytocics in tropi-
cal climates,14 the World Health Organization (WHO)
Additional Supporting Information may be found in the online
version of this article. Supporting Information
Correspondence to: Christian Scho¨neich (Telephone: +785-864-
4880; Fax: +785-864-5736; E-mail: schoneic@ku.edu)
Journal of Pharmaceutical Sciences
© 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
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MOZZICONACCI AND SCHONEICH
2
MSn analysis presents two advantages: (i) to investi-
gate the structure of products, which are not formed
in sufficient amount for Nuclear Magnetic Resonance
(NMR) analysis and (ii) to limit chemical steps to lin-
earize the product prior to analysis.
duction of the disulfide bonds. The samples were in-
cubated at 45◦C for 30 min. Then, 150 :L of NEM
(10 mM) were added to each sample to derivatize the
thiol groups (including residual DTT). The samples
were incubated at 45◦C for 30 min.
Sample Preparation for Capillary Liquid
Chromatography–MS Analysis
MATERIALS AND METHODS
Materials
The samples were diluted (1/10) in milliQ H2O. Sam-
ple (10 :L) was injected onto a Vydac column (25 cm
× 0.5 mm C18, 3.5 :m), and eluted with a linear gra-
dient delivered at the rate of 20 :L/min by a capillary
liquid chromatography (LC) system (Waters Corpo-
ration, Milford, Massachusetts). Mobile phases con-
sisted of water/acetonitrile (ACN)/formic acid at a ra-
tio of 99%, 1%, 0.08% (v/v/v) for solvent A and a ratio
of 1%, 99%, 0.06% (v/v/v) for solvent B. The following
linear gradient was set: 1%–40% of solvent B within
20 min. Electrospray ionization–MS spectra of the
photoproducts were acquired either on a SYNAPT-G2
(Waters Corporation) or an LTQ-FT hybrid lin-
ear quadrupole ion trap Fourier transform ion cy-
clotron resonance (FT-ICR) mass spectrometer (Ther-
moFinnigan, Bremen, Germany).21 The SYNAPT-G2
was operated for maximum resolution with all lenses
optimized on the [M + 2H]2+ ion from the [Glu]1-
fibrinopeptide B. The cone voltage was 40 V and Ar
was admitted to the collision cell. The spectra were
acquired using a mass range of 50–2000 amu. The
data were accumulated for 0.7 s per cycle. Collision-
induced dissociation (CID) of fragment ions, referred
to as MS2 and MS3, was performed on the FT-ICR
(ThermoFinnigan).21 MS3 were acquired with an at-
tenuation of the parent ion in the range of 45%–55%.
The mass window to collect the parent ion was fixed
to 0.2 Da.
Ammonium bicarbonate (NH4HCO3), dithiothreitol
(DTT), N-ethylmaleimide (NEM), and sodium dihy-
drogen and monohydrogen phosphate were supplied
by Sigma–Aldrich (St. Louis, Missouri) at the highest
purity grade. Oxytocin was purchased from Bachem
(Torrance, California). The structure of oxytocin is
presented in Chart 1.
Ammonium bicarbonate buffer was prepared at a
concentration of 50 mM at pH 7.8. Sodium phosphate
buffer was prepared at a concentration of 20 mM at
pH 7.0. The reaction mixtures were either gently
purged with Ar or left under air. UV irradiations
at λ = 253.7 nm and λ ≥ 290 nm were carried out
by means of a photoirradiator (RayonetTM, South-
ern New England, Branford, Connecticut; RMA-500)
equipped with four lamps (for λ = 253.7 nm, RPR-
˚
˚
2500A lamps were used; for λ ≥ 290 nm, RPR-3000A
˚
lamps were used). The RPR-3000A lamps are charac-
terized by a spectral distribution between 250 and
350 nm, with λmax = 300 nm. However, the use of
R
Pyrexꢀ glass for irradiations at λmax = 300 nm ef-
fectively filters out light with λ < 290 nm.20 The total
flux at λ = 253.7 nm was 3.2 × 10−7 einstein/s.
Methods
Photodegradation of Oxytocin
In a first experiment, oxytocin was dissolved to
20 mM phosphate buffer, pH 7.0, at a concentration of
250 :M. The solutions were either placed in quartz or
Sample Preparation for High-Performance Liquid
Chromatography Analysis–UV Detection
R
Pyrexꢀ tubes prior photoirradiation at λ = 253.7 nm
Non irradiated and UV-irradiated samples were an-
alyzed in accordance with the European Pharma-
copeia’s protocol.22 The samples were analyzed by
means of a Shimadzu chromatographer (Shimadzu,
Torrance, California) equipped with two pumps (LC-
20AT; Shimadzu Prominance) delivering a flow rate of
0.9 mL/min, and a diode array detector (SPD-M20A;
Shimadzu Prominance). The chromatographer was
equipped with a Spherisorb column (4.6 × 250 mm,
S5 ODS2; Waters Corporation). The analytes (50 :L
injection volume) were eluted from the column with
two eluents consisting of (i) sodium dihydrogen phos-
phate (0.1 M, solvent A) and (ii) H2O/ACN (75%:25%
(v/v), solvent B). The column was equilibrated with
70%:30% (v/v) of solvents A /B. The solvents were
eluted with a linear gradient over a period of time of
30 min to reach a solvent composition of A:B 40%:60%
(v/v).
or λ ≥ 290 nm under Ar, respectively. In a second pro-
tocol, oxytocin powder provided by Bachem (in its ac-
etate salt) was simply diluted in milliQ H2O to reach
a concentration of 700 :M. The pH was adjusted with
HCl to be 4.0. The samples were photoirradiated at λ
= 253.7 nm or λ ≥ 290 nm in air, respectively.
Thermal Stability of Oxytocin
Oxytocin and oxytocin, which was photoirradiated at
λ ≥ 290 nm, were incubated overnight at 45◦C.
Reduction of Disulfide Bonds and Alkykation of
Products
After photoirradiation and/or incubation at 45◦C,
200 :L of each sample were diluted with 800 :L of
50 mM NH4HCO3 buffer, pH 7.8. Fifty microliters
of DTT (10 mM) were added to each sample for re-
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PHOTODEGRADATION OF OXYTOCIN AND THERMAL STABILITY OF PHOTOPRODUCTS
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Chart 1. Structure of oxytocin.
reported in the European Pharmacopeia.22 The major
peaks were collected and analyzed by MS.
RESULTS
For simplification, the structures of oxytocin (see
below) and its photoproducts are represented in one
letter code, where “S” refers to the sulfur atom (since
the peptide sequence does not contain Ser). The
tentative structures of the photoproducts, assigned
based on MS/MS analysis, are presented in Table 1.
UV Irradiation Under Ar at λ = 253.7 nm
After UV irradiation of oxytocin (700 :M) under Ar at
λ = 253.7 nm, products 1, 2, 3, 4a, and 4b were ob-
served. Analogous products have been detected after
photoirradiation of a small cyclic disulfide-containing
model peptide, for which a thioether, analogous to
product 3, and a dithiohemiacetal, analogous to prod-
uct 2, were supported by two-dimensioanal NMR
analysis.17
UV Irradiation of Oxytocin
Oxytocin (m/z 1007.4) was photoirradiated at λ =
253.7 nm and λ ≥ 290 nm for 30 min in Ar- or air-
saturated solution. The samples irradiated under
Ar were analyzed by LC–MS (Fig. 1). Four major
photoproducts, referred to as 1, 2, 3, and 4, were
identified. The photoproducts were characterized by
LC–MS/MS analysis and are tentatively assigned
to the structures displayed in Table 1. The sam-
ples photoirradiated in air were analyzed by high-
performance liquid chromatography (HPLC) coupled
to UV detection (λ = 230 nm) according to the protocol
Product 1. Product 1 is the result of the dispropor-
tionation of a pair of thiyl radicals into thiol and
thioaldehyde, where the thioaldehyde has hydrolyzed
into aldehyde (see Discussion, Scheme 1). The MS2
spectrum of 1 allows to identify the aldehyde and
the thiol group on the original N-terminal and C-
terminal cysteines, respectively (Fig. 2). The b4, b6,
y3, and y4 fragment ions demonstrate that the C-
terminal cysteine is derivatized with NEM. The b4
fragment ion demonstrates the loss of 18 Da from
the original N-terminal cysteine, consistent with the
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MOZZICONACCI AND SCHONEICH
Table 1. Overview of the Different Products Tentatively Assigned by Mass Spectrometry
Reference
Structure
Reference
Structure
1
4b
2
5
6
3
4a
The free thiol groups were derivatized with NEM. In products 4a and 6, the carbon–carbon double bond is located between the
carbons C and $C of Cys residue.
conversion of the Cys mercaptomethyl side chain into
the aldehyde.
steps. First, the formation of the acylium ion allows
the loss of 28 Da and the formation of the ion with
m/z 928.5 (reaction S1-7). Second, the formation of the
carbon–carbon double bond permits a nucleophilic ad-
dition from any amide nitrogen (reaction S1-7). The
latter creates a protonated amide bond, which can be
cleaved (reaction S1-8) to permit the loss of CO (−28
Da, reaction S1-9) and the formation of the ion with
m/z 900.3. The use of the C-terminal amide group in
reaction S1-7 permits the loss of 57 Da (C2H3NO) in
reaction S1-10, and the formation of the ion with m/z
843.2. In the MS2 spectrum of 2 (Fig. 3), we tenta-
tively assign the ion with m/z 928.5 to the b7 frag-
ment, which lost NH3, that is, b7-NH3. However, the
absence of the native b7 ion suggests that the ion with
m/z 928.5 is more likely related to the fragmentation
of the ion with m/z 956.5 as described above. Notewor-
thy, the ion with m/z 928.5 is part of the combination of
the ions with m/z 956.5, 928.5, 900.2, and 843.2, which
are necessary to explain the fragmentation mecha-
nism depicted in Scheme S1. This series of ions sup-
ports the proposed mechanism presented in Scheme
S1. Moreover, the cyclic structure of the ion with m/z
956.5, as displayed in Scheme S1, is supported by the
fragment ions obtained during its MS3 fragmentation
(Fig. S1). Indeed, MS3 of the ion with m/z 956.5 shows
Product 2. Product 2 corresponds to the transfor-
mation of the disulfide bond into a dithiohemiacetal.
Because of the presence of a non reducible C S bond,
the MS2 spectrum of 2, displayed in Figure 3, shows
mainly the b6–b9 fragment ions. The loss of 159 Da
from the b6 ion (m/z 689.2) is consistent with the loss
of 1-ethyl-3-mercapto-pyrrolidine-2,5-dione, shown in
the insert of Figure 3a. A proposed mechanism lead-
ing to the loss of 159 Da is detailed in Scheme S1
(Supplementary Information), along with the ratio-
nalization of the formation of the fragment ion with
m/z 843.2, displayed in the zoom below the main spec-
trum in Figure 3b. From the parent ion (m/z 1132.5),
the proton is transferred to the sulfur atom connected
to NEM (Scheme S1, reaction S1-1). Such transfer al-
lows the loss of 159 Da (reaction S1-2). The following
addition of an amide to the carbocation permits the
formation of a new mobile proton, which can trans-
fer to the N-terminal amine (reactions S1-3 and S1-
4), allowing the loss of NH3 and the formation of
the ion with m/z 956.5 (reaction S1-5). A subsequent
rearrangement of the tetracyclic intermediate (reac-
tions S1-6 and S1-7) allows to rationalize the next two
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Figure 1. LC–MS analysis of photoirradiated oxytocin at λ = 253.7 nm under Ar. (a) Original
LC–MS chromatogram displaying the different photoproducts. (b–e) Mass search ([M + H])
of the major products resulting from the photoirradiation of oxytocin. Disulfide bonds were
reduced by DTT and derivatized with NEM.
a first series of ions with m/z 882.3, 769.4, and 672.1,
corresponding to the loss of the residues Gly–NH2
(Scheme S2, S2-2), Leu–Gly–NH2 (Scheme S2, S2-3),
and Pro–Leu–Gly–NH2 (Scheme S2, S2-4) from the
parent ion (m/z 956.5). These three fragments are
easily removed from the parent ion since they are
not part of the cyclic structure. Besides, after the loss
of H2S from the parent ion, the ion with m/z 922.4
is at the origin of the formation of the ions with m/z
714.2 and 601.3. Indeed, the difference of mass be-
tween 922.2 and 714.2 Da is 208 Da, which matches
the sum of 163 + 45 Da. Thus, 208 Da is the combina-
tion of the masses of the monoisotopic masses of Tyr
(163 Da) and an amide group (CONH3, 45 Da). The
loss of Tyr should be followed by the loss of Ile since
the latter is adjacent to Tyr in the sequence of the par-
ent ion (Scheme 1). The MS3 analysis (Supplementary
Fig. S1) indicates a difference of mass of 113 Da
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Scheme 1. Simplified reaction scheme summarizing the formation of the major photoproducts
of oxytocin.
between the ions with m/z 714.2 and 601.3. This
loss corresponds to the loss of Ile. The ions with
m/z 714.2 and 601.3 are the result of a fragmenta-
tion occurring after the loss of H2S from the parent
ion. Thus, an opening of the thioether bond must pre-
cede the loss of Tyr and Ile. The mechanism to open
the thioether bond during the MS3 of the parent ion
with m/z 956.5 is likely to be similar to the one that we
will describe below to explain the MS2 fragmentation
of product 3 (Scheme S3, reaction S3-9 to S3-11).
cyclic peptide has opened. The formation of the ion
with m/z 830.5 is the result of the loss of H2S (34 Da).
Thus, we need first to explain the intermediate ion
with m/z 864.4. The latter must result from a loss of
111 Da from the parent ion. Thus, the ion formed af-
ter the reaction S3-3, which is isobaric to the parent
ion, needs to be rearranged to allow further loss of the
fragment with a mass of 111 Da, as described in reac-
tion S3-8. Reactions S3-4 to S3-7 rationalize such re-
arrangement. An analogous rearrangement has been
documented by Wysocki et al.23 The intermediate ion
formed through reaction S3-7 permits the neutral loss
of 111 Da and the formation of the ion with m/z 864.4
(reaction S3-8). The protonated primary amine in the
product ion allows for proton transfer to the sulfur
atom (reaction S3-9). Subsequently, the original N-
terminal opens the thioether bond and regenerates
a free mobile proton (reaction S3-10). The transfer
of the proton from the amine to the thiol group al-
lows the protonation of the thiol function and the loss
of H2S (reactions S3-11 and S3-12) to generate the
ion with m/z 830.5. To support the structure of the
ion with m/z 830.5 as displayed in Supplementary
Scheme S3, this ion was further fragmented in MS3
experiments. The MS3 spectrum of the ion with m/z
830.5 is presented in Supplementary Figure S2. Five
fragment ions with m/z 813.1, 785.4, 717.2, 700.2, and
537.3 are generated. The formation of these ions is
rationalized in Supplementary Scheme S4 (Supple-
mentary Information). The formation of the series of
ions with m/z 813.1, 785.4, and 700.2 corresponds to
Product 3. Product 3 corresponds to the transfor-
mation of the dithiohemiacetal (product 2) into a
thioether.17 The structure of 3 is characterized by
its MS2 spectrum (Fig. 4). The presence of the b6–b9
and y3 fragment ions, as well the internal fragment
YIQ and the absence of the other b fragments pro-
vide evidence for the formation of a thioether bond
between the two cysteine residues. The ion with m/
z 830.5 is rationalized through the mechanism dis-
played in Supplementary Scheme S3 (Supplementary
Information). From the parent ion (m/z 975.4), the mo-
bile proton can be transferred to the amide nitrogen
that belongs to the peptide bond between the residues
Ile and Gln (reaction S3-1). The amide group of the
side chain of Gln can cyclize with the C-terminal side
of Ile (reaction S3-2). At the same time, the primary
amine which resulted from the cleavage of the peptide
bond between Ile and Gln cyclizes with the carbonyl
group of the side chain of Gln (reaction S3-3). So far,
the mass of the ion has not changed, but the original
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Figure 2. CID fragmentation of product 1 obtained on a SYNAPT-G2 mass spectrometer.
Mass spectra at the bottom represent zoom-ins into the main mass spectrum (top).
717.2 is 113 Da and corresponds to the monoisotopic
mass of Leu or Ile. The loss of Ile must be preceded
by the loss of NH3, as it is explained through reac-
tions S4-1 to S4-3. Therefore, the direct loss of 113 Da
from the parent ion cannot match the loss of Ile.
the following fragmentation steps: (i) the loss of NH3
from the parent ion (Scheme S4, reactions S4-1 to S4-
3), (ii) the loss of CO (reaction S4-4), and (iii) the loss
of the 2-methylbutan-1-imine (reaction S4-5). The dif-
ference of mass between the ions with m/z 830.5 and
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Figure 3. CID fragmentation of product 2 obtained on an FT-ICR mass spectrometer. Mass
spectrum at the bottom represents zoom-in into the main mass spectrum (top).
Consequently, the loss of 113 Da from the parent
ion can only be rationalized through the loss of the
monoisotopic mass of Leu. The cyclization of the
amide group of Gly with the carbocation present in
the original parent ion (reaction S4-6) generates a
free mobile proton permitting the protonation of the
amide bond of Gly (reaction S4-7) and the internal
fragmentation (reactions S4-8 and S4-9), which al-
lows the release of Leu. The last fragment ion with
m/z 537.3 (Supplementary Fig. S2) corresponds to the
loss of Tyr–Ile–NH2. This fragmentation is the result
of the protonation of the amide bond of Tyr (reactions
S4-10 and S4-11).
as in product 4b (theoretically, such coupling can also
involve the "C carbons of Cys, but steric reasons likely
prevent such a coupling).
In the MS2 spectrum presented in Figure 5b, the
fragment ions with m/z 513.1, 627.3, 755.2, and 868.3
can be assigned to the y4, y5, y6, and y7 fragment ions
of product 4a. However, their intensities are not com-
parable to the intensities of the same ions observed
during the fragmentation of 5 (see below), suggesting
that structure of 4b rather than 4a is formed. An anal-
ogous product was observed after photoirradiation of
a cyclic disulfide-containing model peptide.17
UV Irradiation Under Air
Products 4a and 4b. Products 4a (Fig. 5a) and 4b
(Fig. 5b) are isobaric. In comparison to product 5 (m/z
1259.5) for which a description will be given below,
4a and 4b correspond to a loss of 2 Da from oxytocin.
The loss of 2 Da can either be explained through the
formation of a vinylic carbon–carbon bond such as in
4a or the formation of a covalent carbon–carbon bond
between the $C carbons of the cysteines residues such
Oxytocin (700 :M) was prepared by diluting the
Bachem powder in milliQ H2O. The pH of the solu-
tion was adjusted to 4.0 by using HCl solution. The
samples were prepared by placing 200 :L of the stock
R
solution in either quartz or Pyrexꢀ tubes (opened to
air) prior to UV irradiation at λ = 253.7 nm or λ ≥
290 nm, respectively.
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Figure 4. CID fragmentation of product 3 obtained on a FT-ICR mass spectrometer. Mass
spectrum at the bottom represents zoom-in into the main mass spectrum (top).
Irradiation at 8 = 253.7 nm. Oxytocin was exposed
to UV light (λ = 253.7 nm) for 0, 5, 15, 30, and
60 min. The chromatograms of the non irradiated
and irradiated samples are presented in Figure 6a.
Native oxytocin is eluted after tr = 14.9 min. Over
the time of irradiation, oxytocin was transformed
into product 3 (thioether), which is eluted at tr =
9.7 min (Fig. 6a, peak 2). Peak 2 was collected for MS
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MOZZICONACCI AND SCHONEICH
Figure 5. CID fragmentation of 4a and 4b obtained on a FT-ICR mass spectrometer. Mass
spectrum at the bottom represents zoom-in of the main mass spectrum (top). “u” refers to the
derivatization of Cys with NEM. The fragment ions in circles are related to the fragmentation
of product 4b. All the other ions are common to both products 4a and 4b.
analysis that revealed a mass-to-charge ratio of 975.5,
The chromatograms of the non irradiated and irra-
diated samples are presented in Figure 6c. Native
oxytocin is eluted after tr = 14.9 min. Over 60 min of
UV exposure, we did not observe the formation of an
additional peak except the one assigned to the native
oxytocin. However, over the time of UV irradiation,
the absorption intensity (monitored at λ = 230 nm)
of peak 1 decreased (Fig. 6c). MS analysis revealed
that the mass-to-charge ratio of the product(s) eluted
at tr = 14.9 min was isobaric to the native oxytocin
(m/z 1007.4, Supplementary Fig. S4b). We therefore
assumed that the decrease of the absorption intensity
of peak 1 over the time of irradiation could be related
to the transformation of oxytocin into its dithiohemi-
acetal derivative (product 2, where the free thiol is not
isobaric to product 3 (Supplementary Fig. S4a).The
relative abundance of oxytocin and product 3 was
monitored over 30 min of irradiation (Fig. 6b). Within
30 min of UV exposure, the amount of oxytocin de-
creased linearly as the amount of product 3 increased.
Over 30 min of UV exposure, oxytocin was completely
transformed into 3. However, a prolonged irradiation
of product 3 in air induced its degradation into un-
known photoproducts, which were eluted between 2
and 8 min.
Irradiation at 8 ≥ 290 nm. Oxytocin was exposed to
UV light (λ ≥ 290 nm) for 0, 5, 15, 30, and 60 min.
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Figure 6. HPLC/UV-detection (8 = 230 nm) analysis of oxytocin. a)Oxytocin was UV-
irradiated at 8 = 253.7 nm in air for 0 min (black), 5 min (red), 15min (blue), 30 min (pink),
60 min (green). b) Monitoring of the conversion of oxytocin (᭹) (peak 1, a) into its thioether
derivative (᭺) (product 3, peak 2, a). c) Oxytocin was UV-irradiated at 8 > 290 nm in air for 0
min (black), 5 min (red), 15 min (blue), 30 min (pink), 60 min (green). d) Monitoring of the trans-
formation of oxytocin (᭹) into its dithiohemiacetal derivative (ꢀ) (product 2 (non derivatized
with NEM), peak 1, c).
derivatized with NEM). To confirm such hypothesis,
the oxytocin sample irradiated for 60 min was deriva-
tized with NEM (Fig. 7). The chromatograms pre-
sented in Figure 7 show that the non irradiated oxy-
tocin is not derivatized with NEM (Figs. 7a and 7b),
where, after 60 min of UV exposure and NEM deriva-
tization, a new peak appeared (tr = 17.8 min, Fig. 7c)
with an absorption approximately 10-fold higher than
the absorption of peak 1 presented in Figure 6c. The
higher intensity of the absorption is likely due to the
conjugation of the maleimide group to the oxytocin
photoproduct. To confirm the addition of NEM to the
photoproduct, the new product eluted at tr = 17.8 min
(peak 2, Fig. 7c) was collected for MS analysis. The
latter revealed that the product is isobaric to product
2 (m/z 1132.5, Supplementary Fig. S4c). Because the
non-irradiated oxytocin was not derivatized by NEM,
we assume that the NEM derivatization with the pho-
toproduct is due to the presence of a free thiol, as in a
dithiohemiacetal structure. Thus, in Figure 6c, the ob-
servation of the decrease of the absorption intensity of
peak 1 over the time of irradiation is likely due to the
transformation of the disulfide bond of oxytocin into a
dithiohemiacetal structure (product 2). The decrease
of the absorption intensity can be rationalized by a
smaller molar extinction coefficient at λ = 230 nm of
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MOZZICONACCI AND SCHONEICH
12
the dithiohemiacetal structure in comparison to the
disulfide bond. The calculation of the absorption of
the mixture of the dithiohemiacetal product and oxy-
tocin can be linearized through Eq. 1 (for 0 min < t <
60 min, light pathway l = 1 cm; see equations below).
At the particular time points, t = 0 min and t = 60 min,
we assume that the measure of the absorption is only
due to either the oxytocin or product 2 (where the free
thiol is not derivatized with NEM), respectively (Eqs.
2 and 3). Through Eqs. 2 and 3, we can calculate the
230
values of the molar extinction coefficients ε1
and
230
230
230
ε2
(ε1
= εdisulfide + εaromatic, ε2
= εdithiohemiacetal
+ εaromatic) at λ = 230 nm for oxytocin and product 2
(where the free thiol is not derivatized with NEM),
respectively. Thus, ε1230(oxytocin) = 360 M/cm, and
ε2230(product 2) = 72 M/cm.1 A combination of Eqs.
1 and 4 allows for the determination of the Eqs. 5
and 6, which control the concentration of product 2
(where the free thiol is not derivatized with NEM) and
oxytocin over the time of irradiation, respectively
(Fig. 6d).
At = g2130[oxytocin]t + g2230[product 2]t
(1)
where
230
230
g2130 = g
= g
+ g
and g230
aromatic
disulfide
2
230
230
aromatic
+ g
dithiohemiacetal
at t = 0 min :
A0 = g2130[oxytocin]0
at t = 60 min
(2)
(3)
(4)
A60 = g2230[product 2]60 = g2230[oxytocin]0
[oxytocin]0 = [oxytocin]t + [product 2]t
For 0 < t < 60 min, :
At
288
[product 2]t = 8.75 10−4
For 0 < t < 60 min, :
−
(5)
(6)
At
288
[oxytocin]t =
− 1.75 10−4
Figure 7. HPLC UV-detection (8 = 230 nm) analysis of
oxytocin. a) Native oxytocin. b) Native oxytocin mixed with
NEM. c) Oxytocin was UV-irradiated at 8 > 290 nm in air
for 60 min and derivatized with NEM.
Thermal Stability of Photoirradiated Oxytocin
Oxytocin was photoirradiated at λ ≥ 290 nm for
30 min in Ar and air. Control oxytocin and photoir-
radiated oxytocin were then incubated overnight at
45◦C. LC–MS analyses of the samples pre-irradiated
in Ar are presented in Figure 8. Three major products,
referred to as 2, 5, and 6, were identified (Table 1).
Thermal Stability After Photoirradiation at λ > 290 nm
Under Ar
1
230
230
The molar extinction coefficients g1
and g2
are deduced
from the absorption read at 8 = 230 nm on the absorption spec-
tra recorded during HPLC analysis. Therefore, at tr = 14.9 min,
the molecular extinction coefficients have been calculated in a sol-
vent composition consisting of 45 mM NaH2PO4 in 72.5%:27.5%
H2O:ACN.
The sample photoirradiated under Ar at λ > 290 nm
was subjected to thermal stability testing. The sample
was maintained in Ar during incubation. The LC–MS
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PHOTODEGRADATION OF OXYTOCIN AND THERMAL STABILITY OF PHOTOPRODUCTS
13
Figure 8. LC-MS analysis of oxytocin a)after DTT reduction and NEM derivatization, b) after
photo-irradiation at 8 > 290nm under Ar following by DTT reduction and NEM derivatization, c)
after photo-irradiation at 8 > 290 nm under Ar and overnight incubation at 45oC in Ar, followed
by DTT reduction and NEM derivatization. Peak eluted at 7 min corresponds to molecules of
DTT derivatized with two molecules of NEM.
analysis revealed the presence after incubation of two
major products referred to as 5 and 6.
tion S5-1 and S5-2). The mobile proton present on the
amine can be transferred to the sulfur, leading to the
formation of a free thiol group and a carbocation (re-
action S5-4). Then, the proton of the alcohol function
can be transferred to the secondary amine creating a
new protonated amide bond (reaction S5-5). The mo-
bile proton present on the nitrogen permits, after its
transfer to the thiol group, the release of H2S (reac-
tions S5-6 and S5-7).
Product 5. Product 5 is independently produced by
reduction of oxytocin with DTT and alkylation of the
free thiols by NEM. The MS2 spectrum of 5 (Supple-
mentary Fig. S3) provides evidence for its linear struc-
ture and the derivatization of the cysteine residues by
NEM. The b2–b9, y4–y7, and the internal fragment
ions QNu and IQNuP characterize the structure of 5
(here, u refers to the residue Cys–NEM). The ion with
m/z 1225.3 needs further explanation. This ion is the
result of a loss of 34 Da, most likely H2S, from the
parent ion (m/z 1259.5). Because the thiol functions
are blocked by derivatization with two maleimide
structures, the loss of H2S needs to be rationalized
(Supplementary Scheme S5). After protonation of the
carbonyl oxygen of NEM, the N-terminal amine can
cyclize with NEM into a hexacyclic structure (reac-
Product 6. Product 6 is observed after photoirradia-
tion of oxytocin at λ > 290 nm, followed by incubation
at 45◦C. Product 6 shows similar MS2 fragmentation
(Fig. 9) as the thioether, product 3. The presence of
the b6 and y3 fragment ions provides evidence for the
cyclization between the cysteine residues. However,
the comparison of the mass-to-charge ratios of the y3,
b6, and b7 fragment ions with those produced dur-
ing the MS2 fragmentation of the thioether, product 3
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14
MOZZICONACCI AND SCHONEICH
Figure 9. CID fragmentation of 6 obtainedon a SYNAPT-G2mass spectrometer.
sis by MS. MS analysis of these samples revealed the
(Fig. 4), show clearly a difference of −2 Da, support-
ing the formation of a vinylic group. An analogous
vinyl–thioether structure was detected upon photoir-
radiation of other cyclic-containing model peptides.17
presence of products with molecular weights higher
than that of oxytocin (Supplementary Fig. S5). No
significant difference was observed in the range of
masses monitored by MS after mixing the stressed
sample with DTT. Thus, the high–molecular-weight
products are non reducible, meaning that no disul-
fide structure has survived the photo and thermal
stresses. The high-molecular-weight species likely in-
volve multiple combinations of modifications, where
preliminary data would be consistent with tyrosine
oxidation, cross-linking of tyrosine oxidation products
with thiol or amine residues, and a possible hydrolytic
cleavage at Pro. Future experiments will be designed
to investigate the nature of these non reducible
cross-links.
Thermal Stability After Photoirradiation at λ> 290 nm
Under Air
The sample photoirradiated in air at λ > 290 nm for
30 min was subjected to thermal stability testing. UV
detection revealed the complete transformation of the
photoproducts and oxytocin into new species coeluted
after 30 min (Fig. 10). The photo- and heat-stressed
sample was subjected to MS analyses in different
ways: (i) the sample was immediately analyzed by MS;
(ii) after heat stress, the sample was mixed with DTT
in NH4HCO3 buffer (pH 7.8, 50 mM) prior to analy-
Figure 10. HPLC UV-detection (λ = 230 nm) analysis of oxytocin after UV-irradiation at λ >
290 nm in air and incubation at 45◦C overnight.
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PHOTODEGRADATION OF OXYTOCIN AND THERMAL STABILITY OF PHOTOPRODUCTS
15
On the basis of our previous experiments with
an intrachain disulfide-containing model peptide
and salmon calcitonin, we know that if the
photoirradiation at λ = 253.7 nm is maintained under
Ar flow, the disulfide bond is exclusively transformed
into a thioether.17 With oxytocin, we made a similar
observation. The photoirradiation of oxytocin at λ =
253.7 nm under Ar flow generates exclusively product
3. An important observation is the temperature–sen-
sitivity of the photoproducts, especially of the dithio-
hemiacetal 2. For practical purposes, that means that
any exposure of oxytocin to elevated temperature may
yield significantly different products when such pep-
tide had been exposed to light prior to storage at ele-
vated temperature.
The studies under Ar atmosphere allowed us to de-
termine critical photoproducts generated during UV
light stress of oxytocin. The structures of the pho-
toproducts generated under air are similar to those
observed under Ar. The major differences observed
between experiments under Ar and air occur during
subsequent heat stress. When the photoproducts are
incubated in the presence of air, we observed the for-
mation of non reducible high–molecular-weight prod-
ucts likely combining different types of modifications
(e.g., oxidation of Tyr, cross-linking, and hydrolytic
cleavage).
DISCUSSION
To assist in the following discussion, a simple reaction
scheme is presented in Scheme 1.
The photodegradation of oxytocin at λ = 253.7 nm
and λ ≥ 290 nm leads to the formation of stable photo-
products identified by MS. The absorption of light by
a disulfide bond (λmax ca. 250 nm) results initially in
the formation of a pair of cysteinyl radicals (CysS•).24
In the singlet state, the pair of CysS• radicals can
recombine to regenerate the disulfide bond. However,
rapid intersystem crossing will efficiently generate a
triplet radical pair of CysS• radicals.25 In the triplet
state, the parallel spins of the CysS• radicals pre-
vent such recombination. Thus, in the triplet state,
the two CysS• radicals will most likely dispropor-
tionate to yield a thiol and a thioaldehyde.26,27 In
H2O, the thioaldehyde is rapidly transformed into
aldehyde, explaining the formation of 1. We demon-
strated with an intrachain disulfide-containing model
peptide and with salmon calcitonin that such dis-
proportionation reaction occurred.17 However, in the
particular case of an intrachain disulfide bond, the
disproportionation products can rapidly convert into
a dithiohemiacetal (product 2).17 In product 2, the
S C S structure is similar to that in 1,3,5-trithiane
and 1,3-dithiane. Steady-state and laser flash pho-
tolysis studies of derivatives of 1,3,5-trithiane have
shown the formation of radical or ionic species
through homolytic or heterolytic cleavages of the C S
bond.28–31 This observation suggests that the struc-
ture S C S in product 2 is photosensitive at λ =
253.7 nm, consistent with our earlier data on the
photosensitivity of dithiohemiacetal of a cyclic model
peptide.17 Thus, the continuous exposure of 2 (where
the free thiol is not derivatized with NEM) at λ =
253.7 nm leads to the formation of product 3. In ad-
dition, we observed that the incubation of the dithio-
hemiacetal photoproduct of oxytocin leads to the for-
mation of product 6. Such observation is consistent
with our previous study where we reported that a
dithiohemiacetal structure is not only photosensitive
but also heat labile.17 Our results show that the HPLC
methodology recommended in the pharmacopeia to
study the stability of oxytocin cannot discriminate na-
tive oxytocin from its dithiohemiacetal derivative.
Our MS2 data do not provide sufficient detail to dis-
tinguish the isobaric structures 4a and 4b. However,
we know that the formation of a biradical molecule
may lead to the formation of product 4b. Indeed,
a formal 1,2-H-shift reaction occurring in each of
the CysS• radicals would generate two $C carbon-
centered radicals, which ultimately would react to-
gether to form a C C bond. The formation of prod-
uct 4b and the observation of a similar structure
during photo-irradiation of an intrachain disulfide-
containing model peptide support the occurrence of
such 1,2-H-shift processes.17
CONCLUSION
Our study shows that at pH 4.0–5.0 or pH 7.0–8.0,
oxytocin is sensitive to UV light. Oxytocin is easily
transformed in presence of Ar or air into its thioether
and dithiohemiacetal derivatives after UV exposure
at λ = 253.7 nm and λ > 290 nm, respectively. Under
inert atmosphere, the combination of light and heat
stresses leads to the transformation of the disulfide
bond of oxytocin into a thioether. Under air, subse-
quent heat stress leads to the formation of non re-
ducible cross-links.
ACKNOWLEDGMENTS
We gratefully acknowledge financial support from
Amgen Inc.
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