And Cystine-Containing Cyclic Peptides: Octreotide and Somatostatin

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The effect of conformation on the photo-degradation of Trp- and cystine-containing cyclic peptides: octreotide and somatostatin. Olivier Mozziconacci, and Christian Schoneich Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5003174 • Publication Date (Web): 26 Aug 2014 Downloaded from http://pubs.acs.org on August 31, 2014

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Molecular Pharmaceutics

The effect of conformation on the photo-degradation of Trp- and cystine-containing cyclic peptides: octreotide and somatostatin.

Olivier Mozziconacci☥, and Christian Schöneich☥,* ☥

Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue,

Lawrence, Kansas 66047 * Correspondent author: Christian Schöneich, Tel: 785-464-4826, Fax: 785-864-5736, Email: [email protected]

Keywords: Somatostatin, methyleneindolenine, disulfide.

octreotide,

photochemistry,

ACS Paragon Plus Environment

stability,

tryptophan,

3-

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Abstract The synthetic cyclic octapeptide octreotide is less stable under UV and fluorescent lights than

its naturally occurring derivative somatostatin. Upon irradiation at λ > 290 nm, and in the presence of oxygen, octreotide is quickly transformed into a photoproduct showing an increase of mass of 16 Da. The increase of 16 Da to the mass of octreotide is related to a complex transformation where i) the side chain of D-Trp is transferred onto the side chain of the Lys residue and ii) the former D-Trp residue is transformed into hydroxyglycine, which ultimately fragments. The complex sequence of photo-transformation is orchestrated by the presence of DTrp and the unusual orientation of its carbonyl group toward the disulfide bond. These transformations are summarized in Scheme 1. The photo-transformation of octreotide is illustrated by product A (Scheme 1, Figure 1).


2

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1


2

Introduction

2

Synthetic peptides have become an important class of drugs since the development of solid-

3

phase peptide synthesis,1 which permits the generation of any primary sequence composed of

4

amino acid residues in their absolute L- or D-configurations.

5

Many peptide hormones have found use as therapeutic agents such as, e.g. arginine

6

vasopressin,2-6

oxytocin,7-11

7

calcitonin,19-24 and somatostatin (Chart 1).25-30 Analogs of these peptides have been developed to

8

subdue certain limitations of the naturally occurring peptides, such as the stability against

9

proteases, and the binding to receptors, to ultimately improve drug action. These analogs often

10

contain D-amino acids. For example, a large number of D-analogs of somatostatin, e.g., D-

11

Cys[14]-somatostatin, and D-Trp[8]-somatostatin (Chart 1), have been synthesized to inhibit

12

more efficiently (than insulin) the secretion of growth hormone and glucagon,31,32 or retain high

13

overall activity.33

14

disorders, has a limited in vivo half-life of 1-2 min.34 Extensive studies on somatostatin have

15

shown that the sequence L-Phe-L-Trp-L-Lys-L-Thr is essential for biological activity, and that

16

the activity can be increased by replacing L-Trp by its enantiomer, D-Trp.33 A search for the

17

most stable in vivo somatostatin analog concentrated, therefore, on cyclic peptides containing the

18

sequence L-Phe-D-Trp-L-Lys-L-Thr, where the most promising candidate was octreotide (Chart

19

1, Figure 1A). In octreotide, the side-chain of D-Phe[1] occupies a position similar in space as

20

Phe[6] in somatostatin, and protects the disulfide bond against enzymatic attack. In addition, the

21

C-terminal threoninol group renders octreotide more stable against enzymatic degradation35 The

22

overall activity of octreotide is similar to that of somatostatin but octreotide is more selective in

23

the inhibition of growth hormone secretion as compared to the inhibition of insulin secretion.

24

The development of octreotide is a successful story for medical applications36 since it is used in

luteinizing

hormone,12-14

adrenocorticotropic

hormone,15-18

Somatostatin which is employed in the treatment of acute gastrointestinal


3


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1

the treatment of acromegaly,37 the carcinoid syndrome,38 various endocrine tumors,39

2

gastrointestinal motility,40 pancreatic damage,41 and advanced breast and prostate cancer.42

3

Octreotide is often administered by injection. However, in palliative care, to avoid repeated 43

4

injections, octreotide is delivered to patients by continuous subcutaneous infusion.

5

non-refrigerated solutions of octreotide are exposed to light for hours or days. Under such

6

conditions, the photostability of octreotide needs to be reevaluated. Trp is a photosensitive amino

7

acid which can undergo photo-induced electron transfer reactions.44-47 For example, disulfide

8

bonds can be reduced to thiol and thiyl radical through one-electron reduction reactions from

9

both an excited singlet and triplet state of Trp*.48-51 In fact, close distances between Trp and

10

disulfide bonds are responsible for enhanced photosensitivity and a number of photo-degradation

11

products in several proteins.52,53 Especially in octreotide, the presence of D-Trp leads to a

12

particular orientation with regard to the disulfide bond:

13

octreotide54 (Figure 2A) reveals that the carbonyl group of D-Trp points towards the disulfide

14

bond with an O—S distance of 4.5 Å (Figure 2A). The proximity of the Trp carbonyl group to

15

the disulfide bond would facilitate a subsequent electron transfer to the disulfide bond. In

16

contrast to octreotide, the O—S distance between the Trp carbonyl group and the disulfide bond

17

is 7.8 Å in somatostatin (Figure 2B). Hence, we expect significant differences in the photo-

18

degradation of octreotide and somatostatin. In fact, the present paper will demonstrate that the

19

exposure of octreotide to UV and cool white light leads to i) the cleavage of the side-chain of Trp

20

and the subsequent addition of the photoproduct, 3-methyleneindolenine (3-MEI), onto a lysine

21

residue (product A, m/z 518.23 (z=2), Figure 1B, Scheme 1), and ii) the transformation of the

22

original Trp into hydroxyglycine, which ultimately fragments (Scheme 1). We have recently

23

documented the photo-induced transformation of Trp to Gly and Gly-hydroperoxide in IgG1.55

4

Therefore,

the three-dimensional structure of


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1

The current results focus on the effect of peptide conformation on Trp photo-degradation via this

2

pathway, identify additional photoproducts, including the highly electrophilic 3-MEI, and

3

provide mechanistic details on the side chain cleavage.

4 5 6

3

7

Octreotide and somatostatin were supplied by Bachem (Torrance, CA, USA). Ammonium

8

acetate, potassium phosphate dibasic (K2HPO4), sodium phosphate monobasic (NaH2PO4),

9

ammonium bicarbonate (NH4HCO3), bis-(2-mercaptoethyl) sulfone (BMS), dichloromethane

10

(CH2Cl2), and N-ethylmaleimide (NEM) were supplied by Sigma-Aldrich (St Louis, MO, USA)

11

at the highest purity grade. Trypsin at sequencing grade was supplied by Promega (Madison, WI,

12

USA).

Materials

13 14

4

Methods

15 16

4.1

Photo-irradiation

17

Octreotide (210 µM) and somatostatin (210 µM) were prepared in ammonium acetate buffer (20

18

mM, pH 6.9). The air-saturated solutions were placed in pyrex tubes for irradiation with λmax=

19

305 nm. Photo-irradiation was performed by means of an UV-irradiator (Rayonet®, The Southern

20

New England Ultraviolet Company, Branford, CN) equipped with four UV-lamps emitting at

21

λmax= 305 nm (RMR-300Å, The Southern New England Ultraviolet Company, Branford, CN),

22

delivering a power of approximately 5 W/cm2. The lamps emitting at λmax= 305 nm have an

23

emission spectrum range between 280 nm and 330 nm. Thus, the samples were placed in pyrex

24

tubes, which have a cut-off at 290 nm, to avoid the absorption of the photons with λ < 290 nm.

25

Some samples were also exposed to cool white fluorescent light for 10 days, placed at 20 cm


5


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1

distance from a fluorescent light tube (T8 lamp, 30 W, F30T8-CW cool white, General Electric,

2

Fairfield, CN, USA. Spectrum in Supplementary Information Chart S1), delivering a power of

3

0.4 W/cm2. The power of the fluorescent light tube was measured by means of a radiometer JX1-

4

1 (Jelight Company Inc., Irvine, CA).

5 6

4.2

Trypsin digestion

7

Controls and photo-irradiated samples were incubated at 45oC with 2 mM BMS for 30 min. The

8

reduced cysteines were alkylated at 45oC with 5 mM iodoacetamide (IAA). After 1 hour of

9

incubation at 45oC, the samples were purified through an Amicon Ultra™ membrane with a cut-

10

off of 10kDa (Millipore, Billerica, MA, USA). Each sample was placed in an Amicon Ultra™

11

tube of which the membrane was equilibrated with ammonium bicarbonate buffer (NH4HCO3,

12

50 mM, pH 8.0). After purification, for each sample a volume of ~20 µL was recovered and

13

diluted with 480 µL of the ammonium bicarbonate buffer. The samples were then incubated at

14

37oC for 1 hour in the presence of 2 µg trypsin. After one hour, an additional 10 µg of trypsin

15

was added to each sample and the incubation continued at 37oC for additional 2 hours. The

16

digestion was stopped by the addition of 20 µL of formic acid (10% in water, v:v) to each

17

sample. The tryptic peptides of somatostatin and octreotide are presented in Chart 1.

18 19 20

4.3

Mass spectrometry analysis

21

The samples photo-irradiated in the photo-irradiator (Rayonet) were analyzed on a Micromass Q-

22

TOF Premier (Waters Corp., Milford, MA, USA) mass spectrometer. A volume of 5 µL of the

23

digests was injected onto a reverse-phase capillary C18 column (Vydac, 250 x 0.5 mm, 5 µm)

24

which was attached to a capillary set of pumps (CapLC), delivering a mixture of solvents A

6


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1

(99% H2O, 1% acetonitrile (ACN), 0.1% formic acid (FA), v:v:v) and B (80% ACN, 10% H2O,

2

10% isopropanol (iPr), 0.1% FA, v:v:v) at a flow rate of 20 µL/min. A gradient of solvents A

3

and B was delivered according to the following program: between 0 and 1 min, the mixture of

4

solvents consisted of 99% solvent A and 1% solvent B. After 1 min, the content of solvent B was

5

increased linearly to 60% within 34 min.

6

The samples exposed to cool white fluorescent light were analyzed on an LTQ-FT mass

7

spectrometer (Thermo Finnigan, West Palm Beach, FL, USA) under conditions described

8

elsewhere.56 The peptides were separated on a reverse-phase LC Packings PepMap C18 column

9

(0.300 × 150 mm) at a flow rate of 10 µL/min with a linear gradient rising from 0 to 65%

10

acetonitrile in 0.06% aqueous formic acid over a period of 55 min using an LC Packings

11

Ultimate Chromatograph (Dionex, Thermo Scientific, Sunnyvale, CA, USA).

12

experiments were performed in a data-dependent acquisition mode using the Xcalibur 2.0

13

software (Thermo Scientific, Sunnyvale, CA, USA). The five most intense precursor ions in a

14

survey MS1 mass spectrum acquired in the Fourier transform-ion cyclotron resonance over a

15

mass range of 300–2000 m/z were selected and fragmented in the linear ion trap by collision-

16

induced dissociation (CID). The ion selection threshold was 500 counts.

LC-MS

17 18

5

Results

19 20

The results presented below will describe how the photo-irradiation of octreotide leads to the

21

formation of product A (Figure 1B, Scheme 1) of which the increase of 16 Da to the mass of

22

octreotide is related to a complex transformation where i) the side chain of D-Trp is transferred

23

onto the side chain of the Lys residue and ii) the former D-Trp residue is transformed into

24

hydroxyglycine, which ultimately fragments.


7


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1 2

5.1

Photo-irradiation of somatostatin

3 5.1.1

4

Irradiation at λmax = 305 nm

5

The LC-MS analyses of tryptic digests of somatostatin obtained before (control) and after 20 min

6

of photo-irradiation at λmax= 305 nm do not show any significant difference (Figure 3).

7 8

5.1.2

Irradiation under fluorescent light

9

After 10 days of exposure to fluorescent light, the solution of somatostatin develops a yellow

10

color. The LC-MS analysis of degraded somatostatin reveals the formation of a series of

11

oxidation products, originating from Trp, of which the major product is N-formylkynurenine

12

(NFK). The MS/MS spectrum of a tryptic digest of this major photoproduct obtained from

13

somatostatin is presented in the Supplementary Information (Figure S1). The transformation of

14

Trp into NFK is clearly demonstrated by the presence of a series of fragment ions b2-b4 and y2-

15

y4. The oxidation yield of NFK corresponds to 0.08% of the total amount of material recovered

16

for LC-MS analysisa. As NFK was the only product observed, we concluded that only 0.08% of

17

the native somatostatin was converted into its NFK product. Hence, a 10 day exposure to cool

18

white light results in degradation of somatostatin.

19

5.2

Photo-irradiation of octreotide

20 21

5.2.1

Irradiation at λmax = 305 nm

22

After 20 min of photo-irradiation at λmax= 305 nm in air, the LC-MS analysis of a tryptic digest

23

(Figure 4) of octreotide reveals the formation of products 1 and 2, eluted at tel = 14.8 min and tel

a

The yields are calculated as follows: Ip / ∑ Ii, where Ip and ∑ Ii represent the intensity of the product ion, and the sum of the intensities of each individual ion, respectively. These calculations assume that the response factor of each ion is identical. The sum of each individual ion is constant since it represents the different components of the native peptide i.e. the non-oxidized and oxidized tryptic peptides. Therefore, the ratios presented in the text are relative to the total amount of native peptide, which is transformed into the pth product of oxidation.

8


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1

= 10.5 min, respectively (Figure 4A, 3B). When the disulfide bond of octreotide was reduced and

2

the thiols of the reduced cysteines were alkylated prior to irradiation, we did not observe the

3

formation of any products (Figure 4C), indicating the importance of an intact disulfide for

4

photoproduct formation. Irradiation of octreotide in presence of Ar did not produce any

5

photoproducts (Figure 4D). The mass-to-charge ratios (m/z) of 1 and 2 are m/z 871.4 and m/z

6

540.4, respectively (Scheme 1). Product 1 corresponds to a modification occurring within the

7

tryptic peptide T’1 of octreotide (Chart 1), where 3-MEI is lost from Trp and transferred onto the

8

side-chain of the Lys residue (Scheme 1). Concomitant with the transfer of 3-MEI to the Lys

9

residue, the loss of the side-chain of D-Trp results in the transformation of D-Trp into

10

hydroxyglycine (Scheme 1). Subsequently hydroxyglycine converts into product 2. After 20

11

minutes of photo-irradiation, the yields of 1 and 2 correspond to 1.6% and 14% of the total

12

amount of material recovered during the LC-MS analyses,a respectively. Therefore, we

13

concluded that 15.6% of native octreotide was photodegraded after 20 min of irradiation in air at

14

λmax = 305 nm. The observation of stable hydroxyglycine in peptide structures has been reported

15

for natural products.57,58 Products 1 and 2 are characterized by their MS/MS fragmentation of

16

which a detailed description is given below. Product 3 could not be detected. The presence of an

17

aldehyde function in 3 (Scheme 1) likely promotes further reaction of 3 with any primary amine

18

present in either the native octreotide or the photoproducts. Over prolonged photo-irradiation at

19

λmax=305 nm for 90 minutes, we observe that the ion intensity of 1 decreases (Figure 5A), while

20

that of product 2 increases linearly (Figure 5B). This observation suggests that 2 originates from

21

product 1.

22

The collision induced dissociation (CID) spectrum of the ion with m/z 871.4 allows for the

23

identification of the sequence of product 1 (Figure 6). Indeed, the presence of the fragment ions


9


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1

b2 and y3-y4 demonstrate that Cys is derivatized with NEM. The fragment ions b3-b4 and y2

2

validate the transformation of Trp into hydroxyglycine and the migration of 3-MEI onto the side-

3

chain of Lys. The CID spectrum of the ion with m/z 540.4 permits the identification of the

4

sequence of product 2 (Figure 7). The b2 and y2 fragment ions demonstrate the presence of Cys

5

derivatized with NEM and the presence of Phe residues. The absence of loss of ammonia from

6

the fragment ion b2 and the abundance (50% of the total ion counts) of the fragment ion with m/z

7

523.1, which corresponds to the neutral loss of ammonia from the parent ion, reinforce the

8

hypothesis of an amidated C-terminal Phe residue.

9

The exposure of aqueous Trp to UV-light can lead to photo-ionization, generating a Trp radical

10

cation, Trp+●, and a hydrated electron (e-aq).46 Hydrated electrons are highly reactive, so that we

11

needed to test whether the formation of either 1 or 2 resulted from reactions of e-aq. Therefore,

12

we photo-irradiated octreotide in the presence of an electron scavenger (dichloromethane,

13

CH2Cl2) at sufficiently high concentration (20 mM) to scavenge 98% of the solvated electrons (e-

14

aq),

15

(k=6.0 x 109 M-1 s-1).60 According to these rate constants and the concentrations of CH2Cl2 (20

16

mM) and disulfides (210 µM, concentration of octreotide), the pseudo first order rate constants

17

of e-aq with CH2Cl2 and disulfide bond are 120 x 106 s-1 and 2.1 x 106 s-1, respectively. The

18

amount of products 1 and 2 generated after 30 min of irradiation in the presence and absence of

19

CH2Cl2 was nearly identical (Figures 4, A and B), suggesting that e-aq is not involved in the

20

formation of products 1 and 2.

21

In order to test whether 3-MEI is released into solution, we photo-irradiated 200 µM octreotide

22

in the presence of 1 mM L-Lys. We observed that products 1 and 2 are formed where the yields

23

of 1 and 2 correspond to 1.5% and 12% of the total amount of material recovered during the LC-

10

based on the rate constants of e-aq with a disulfide bond (k=1.1 x 1010 M-1 s-1)59 and CH2Cl2


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1

MS analyses, respectively, when octreotide was photo-oxidized in the presence of exogenous

2

Lys (Figure S2). This result suggests that the ߜ-NH2 group of the Lys residue reacts with the

3

nascent 3-MEI via nucleophilic substitution before 3-MEI can be released from the Trp+● radical

4

cation.

5 6 7 8

5.2.2

Irradiation under fluorescent light

9

After 10 days exposure of an octreotide solution to cool white fluorescent light, the solution

10

remained transparent. However, the two major oxidation products 1 and 2 were detected. The

11

MS/MS spectra of 1 and 2 generated during the exposure of octreotide under fluorescent cool

12

white light show the same fragmentation pattern as the spectra of 1 and 2, respectively, obtained

13

when octreotide was irradiated for 20 min with λmax= 305 nm (Supplementary Information,

14

Figure S3 and Figure S4).The yields of 1 and 2 observed after 10 days of irradiation of octreotide

15

with cool white fluorescent light correspond to 0.08% and 0.6%, respectively, of the total amount

16

of material recovered during the LC-MS analyses. Hence, with close to 1% oxidation yields

17

during a 10 days exposure, these product yields are significant.

18 19

5.3

Three dimensional structures

20 21

5.3.1

Structures of somatostatin and octreotide

22

A structure of somatostatin was resolved by high-resolution NMR and semi-empirical

23

calculations.61 The crystal structure of octreotide was determined by Pohl et al.54 The structures

24

of somatostatin and octreotide are represented in Figure 2, respectively. In somatostatin, the


11


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1

distance between the oxygen atom of the carbonyl group of the L-Trp residue and the disulfide

2

bond is 7.8 Å. The shorter ring structure of the cyclic peptide and the presence of D-Trp in

3

octreotide significantly shortens the distance between the oxygen atom of the carbonyl group of

4

D-Trp and the disulfide bond to 4.5 Å.b In addition, in octreotide the proximity of D-Trp and

5

cystine creates a hydrogen bond between the amide of the C-terminal Cys residue and the

6

carbonyl group of D-Trp (distance (C=O)D-Trp-(NH)Cys-C-term = 1.8 Å).

7

6

Page 12 of 34

Discussion

8

An important result from our product study is that the natural hormone somatostatin and its

9

synthetic equivalent, octreotide, behave differently upon UV-light exposure. Two different

10

lamps, one emitting at 305 nm and a second one with λ > 350 nm (cool white light) have been

11

used in this study to investigate how surrounding light can affect the stability of peptide drugs.

12

While somatostatin does not degrade significantly after exposure for 20 min to UV-light with

13

λmax = 305 nm, octreotide rapidly undergoes a photochemical reaction allowing for the

14

simultaneous transformation of Trp into hydroxyglycine and the transfer of 3-MEI (the original

15

side-chain of Trp) onto the side-chain of Lys to yield product A, which after reduction and

16

alkylation of the reduced cysteine residue and digestion leads to the chromatographically

17

separable product 1. The formation of 1 is observed within the first minutes of irradiation (Figure

18

5A). Over the time of irradiation, product 1 is transformed into 2. Indeed, over 90 min of UV-

19

irradiation, the yield of 1 decreased by ca. 40% while the amount of 2 increased by almost 4-fold

20

(Figure 5A). A comparison of the three dimensional structures of i) octreotide (Figure 2A), and

21

ii) somatosatin (Figure 2B), shows that the presence of D-Trp brings the carbonyl group (C=O)

22

of D-Trp closer to the disulfide bond. Thus, the C=O function of D-Trp in octreotide (Figure 2A)

23

is perfectly oriented towards the disulfide bond to permit an intramolecular electron transfer b

The crystallographic data of octreotide were obtained with a full resolution range of 0.246-1.063 Å over 18951 observations.

12


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1

from C=O to the disulfide bond. A tentative mechanism for the formation of product 1 is

2

suggested in the following. The photo-excitation of Trp is known to generate an excited state of

3

Trp (Trp*) from which an electron can be ejected. Based on our experimental results, no

4

hydrated electron is involved in the formation of both 1 and 2, confirmed by the addition of an

5

electron scavenger (CH2Cl2) to the solution, which does not significantly change the yields of

6

both 1 and 2 during the irradiation of octreotide. We, therefore, hypothesize that photo-excitation

7

of Trp leads to an intramolecular electron transfer to the C=O function of Trp (Scheme 2,

8

reactions 1 and 2), followed by one-electron reduction of the disulfide bond into the disulfide

9

radical anion (Scheme 2, reaction 3). This mechanism is fully supported by calculations on

10

primary photo processes of Trp, which demonstrate initial electron transfer to the Trp carbonyl

11

function, as well as experimental findings for several disulfide-containing proteins.62-64

12

In peptides and proteins containing Trp, often the lowest unoccupied molecular orbital is

13

associated with the C=O group of the indole. Because electron transfer occurs from the highest

14

occupied molecular orbital of the donor (the indole group of Trp*) to the lowest unoccupied

15

molecular orbital of the acceptor, the C=O function of the peptide bond is, therefore, the most

16

probable electron acceptor.65-69 The electron transfer rate is also a function of distance and

17

orientation of the donor and acceptor groups.70,71 Thus, when the molecular orbitals of a strong

18

quencher such as the disulfide bond, which is known to quench Trp fluorescence by excited-state

19

electron transfer,72 overlap with those of C=O, the final acceptor of the electron transfer can be

20

the disulfide, which is transformed into a disulfide radical anion (SS●-). Near UV irradiation of

21

Trp residues leads to the reduction of nearby disulfide bonds in Fusarium solani psi and goat

22

alpha-lactalbumin.48,73-76 Also, spatial preferences between aromatic residues and disulfide bonds

23

in the geometry of proteins were observed in a series of proteases and immunoglobulins.52,77-79


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

When the disulfide bond of octreotide was reduced and the thiol of the reduced cysteine residues

2

were alkylated prior to irradiation in air, our LC-MS analysis did not reveal the presence of

3

photoproducts (Figure 4C), suggesting that the disulfide is critical the reaction. In peptides and

4

proteins, aromatic and sulfur-containing aliphatic amino acids are relays for intramolecular

5

electron transfer reactions.80 The disulfide bond could therefore act as a relay in the formation of

6

product A.

Page 14 of 34

7

Simultaneously to the formation of SS●-, the formation of the radical cation centered on the

8

nitrogen of the indole group (Scheme 2, reaction 2) allows for the cleavage of the Cα-Cβ bond of

9

Trp+● (Scheme 2, reaction 3).55 However, based on our trapping experiments with added L-Lys,

10

we conclude that the orientation and the proximity of Lys to D-Trp in octreotide results in an

11

efficient intramolecular transfer of 3-MEI to the Lys residue (Scheme 2, reactions 3-4)

12

generating a carbon centered radical on αC (αC●). This study will oblige us to revisit in the near

13

future our IgG1 study,55 since we did not investigate at that time the possibility for the transfer of

14

3MEI to adjacent Lys residues. Only a model such as octreotide could be used at first to

15

highlight the possibility of intramolecular migration of 3MEI.

16

This entire reaction sequence may be viewed as involving a nucleophilic attack of the Lys

17

side chain amino group at the βC-carbon of the D-Trp side chain, which permits the formation of

18

an αC● radical and the transfer of 3-MEI onto the side-chain of Lys (Scheme 2, reaction 4).

19

Parallel to the cleavage of the D-Trp side chain, the disulfide radical anion (SS●-) transfers an

20

electron to O2, generating superoxide radical anion (O2●-). Superoxide can ultimately react with

21

the αC● radical, resulting from the cleavage of the Cα-Cβ bond of Trp (Scheme 2, reaction 5), to

22

generate a hydroperoxide (Scheme 2, reaction 6). The reactions 4-6 were detailed to explain the

23

migration of 3MEI. However, the formation of the biradical intermediate (reaction 4) would

14


Page 15 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

require an immediate rearrangement due to the proximity of the radical sites. Thus, the reactions

2

4-6 are most likely to be concerted. To quantify the presence of hydroperoxide (ROOH), we

3

added catalase to the irradiated octreotide solution to remove H2O2, and analyzed the presence of

4

ROOH with the FOX2 assay.81,82 After 20 min of irradiation at λmax = 305 nm in the presence of

5

air of a solution containing 210 µM of octreotide, 19.8 µM of hydroperoxide were measured by

6

the FOX2 assay. The products 1 and 2, which were obtained after reduction, alkylation, and

7

digestion of the photoproduct A of octreotide, represented approximately 15.6% (i.e 32.8 µM) of

8

the native octreotide.c These results sustain that product A originated from the formation of

9

ROOH through either the addition of superoxide radical to the carbon centered radical (reactions

10

5-6, Scheme 2) or through the addition of O2 to the carbon-centered radical (reaction 10, Scheme

11

3). In our earlier experiments on the photo-irradiation of IgG1, a hydroperoxide product was

12

isolated while for octreotide we succeeded only to isolate the formal reduction product, i.e. the

13

hydroxyglycine product 1, because the reduction of the disulfide bond and the alkylation of the

14

reduced cysteines were performed prior to digestion. An alternative route to product 1 would be

15

oxygen addition to the αC● radical, followed by elimitation of superoxide and the addition of HO-

16

(Scheme 3, reactions 10-12). If the samples were Ar-saturated prior UV-irradiation, product 1

17

was not observed, sustaining that 1 emerged from the reaction of either superoxide radical or

18

molecular oxygen with the carbon-centered radical at Trp (Figure 4D). No dimer was observed

19

after either irradiation in the presence of air or Ar. Interestingly hydroxyglycine has been

20

reported to be rather stable in several natural products.57,58 The subsequent fragmentation of

21

hydroxyglycine occurs during the reduction, alkylation and digestion (Scheme 2, reactions 7-8).

22

The other product resulting from the fragmentation of product 1 should be product 3 (Scheme 2). c

The FOX2 assay of the non-irradiated octreotide solution did not reveal the formation of ROOH. The FOX2 assay of the irradiated solution of octreotide in the presence of Ar revealed the presence of 1.5 µM of ROOH. The assay was calibrated using different concentrations of H2O2. Therefore, the yields of organic hydroperoxides (ROOH) are given as equivalents of H2O2.


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

However, we failed to detect 3, possibly because of its aldehyde group, which may react with

2

any primary amine present in the solution (Scheme 2, reaction 9). Interestingly, the tryptic

3

digestion to yield product 1 appeared to be not affected by the derivatization of Lys with 3-MEI.

4

This is likely due to the fact that Lys derivatization with 3-MEI still allows for protonation of the

5

secondary amine in the final product.83

6

Based on a comparison with the mass spectrometric signal for octreotide, we calculated that

7

1 and 2 represent major photoproducts with yields of 1.6% and 14.0%, respectively, relative to

8

the total amount of material recovered during LC-MS analysis. Importantly, both products 1 and

9

2 were formed when octreotide was exposed to cool white fluorescent light for 10 days. Under

10

these conditions, the yields of 1 and 2 were 0.08% and 0.6%, respectively, relative to the total

11

amount of material recovered during LC-MS analysis. The ratio of 1:2 (0.08:0.6 = 0.13) was

12

approximately the same than that observed after photo-irradiation of octreotide with λmax=305

13

nm (1.6/14 = 0.11).

14

Our results are of mechanistic relevance for analogous processes in proteins such as IgG1. In

15

view of our results, the alkylation of nucleophilic amino acid residues by 3-MEI, derived from

16

Trp photo-oxidation, should be included in any search for potential modifications of proteins

17

exposed to light.

18 19

7

20

We have shown that during the exposure of octreotide to light, the specific conformation,

21

bringing Trp and the disulfide bond in close proximity, causes electron transfer followed by D-

22

Trp side chain cleavage. Such a reaction is not observed for somatostatin, where the L-Trp

23

residue and the disulfide bond are separated over a larger distance.

16

Conclusion


Page 16 of 34

Page 17 of 34

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Chart 1. Sequences of somatostatin and octreotide. The labels T1, T2, T3, T’1, and T’2 refer to the tryptic peptides of somatostatin and octreotide, respectively. The dashed lines indicate tryptic cleavage sites.


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Figure 1. A) Mass spectrum of octreotide prior irradiation (m/z 510.23, z=2). B) Mass spectrum of octreotide after irradiation in air at λ = 305 nm. The product of oxidation of octreotide (product A) has a m/z with 518.23 (z=2).


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Figure 2. A) Structure of octreotide. The coordinates used to build the structure are those reported by Pohl et al.54 B) Structure of somatostatin. The coordinates used to build the structure are those reported by Knappenberg et al.61


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Figure 3. LC-MS analysis of the tryptic digest of somatostatin before (A) and after (B) irradiation at λmax= 305 nm for 20 min. T1, T2, and T3 represent the tryptic peptides of somatostatin presented in Chart 1.


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Figure 4. LC-MS analysis of the tryptic digest of octreotide before (A) and after (B) irradiation at λmax= 305 nm for 20 min. C) LC-MS analysis of the tryptic digest of octreotide where the disulfide bond of octreotide was reduced and the thiol residues of reduced cysteines were alkylated prior to irradiation. D) LC-MS analysis of the tryptic digest of octreotide obtain after irradiation of octreotide in Ar. T’1 and T’2 represent the tryptic peptides of octreotide presented in Chart 1. The peaks colored in blue and red correspond to products 1 and 2, respectively.


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Figure 5. Time dependency of the formation of 1 (A,●) and 2 (B,●) upon photo-irradiation of octreotide at λmax= 305 nm. Error bars are indicated in red. ∆ indicates the amount of 1 and 2 observed when 20 mM CH2Cl2 is present during the photo-irradiation of octreotide. Error bars are indicated in blue.


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Figure 6. CID spectrum of product 1.


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Figure 7. CID spectrum of product 2.


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Scheme 1. Summary of the mechanism of photodegradation of octreotide during exposure to UV-light (λmax= 305 nm).


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Scheme 2. Mechanism of photodegradation of octreotide during exposure to UV-light (λmax= 305 nm).


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Scheme 3. Alternative mechanism of photodegradation of octreotide during exposure to UVlight (λmax= 305 nm).


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Table of Content


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And Cystine-Containing Cyclic Peptides: Octreotide and Somatostatin

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