script bioorganic-lab course · bioorganic-lab course solid phase peptide synthesis 2 table of...

Modul Bioorganische Chemie 11-BCH-0701; 11-Bio-0701; 11-121-1116

Modulverantwortung: Prof. Dr. Annette Beck-Sickinger, Institut für Biochemie,

Brüderstr. 34, 04103 Leipzig; email: [email protected]

1

Script

Bioorganic-Lab Course

3.12.2019 - 20.12.2018

6.1.2020 – 24.1.2020

Saal 6, 3. Etage, Brüderstr. 34, 04103 Leipzig

person in charge:

Sylvia Els-Heindl

email: [email protected]

Bioorganic-Lab Course Solid Phase Peptide Synthesis

2

Table of Contents

Introduction to Solid Phase Peptide Synthesis (SPPS) 3

Block I – Resins and Linkers 5

Block II – Coupling of Amino Acids 11

Block III – Synthesis / Elongation of a Peptide in a Synthesis Robot 17

Block IV – Peptide Analytics 19

Block V – Peptide Modifications on Solid Support 25

References 32

GHS hazard statement 33


3

Introduction to Solid Phase Peptide Synthesis (SPPS)

Peptides gain more and more attention in the development of drugs and thus, synthetic

routes to obtain peptides are of great interest. As therapeutics, peptides have distinct

advantages compared to small molecules or antibody-based drugs as their high affinity

and selectivity. However, they have a low bioavailability due to rapid clearance from

the body and their generally low metabolic stability. To overcome these problems,

peptides can be modified with fatty acids or poly(ethylene glycol), D-amino acids or

acetylation/amidation. Notably, this cannot be accomplished by molecular biology, but

synthetic approaches.[1,2]

Since Emil Fischer discovered peptide synthesis in the early 20th century,[3] the

methods were continually improved. In 1963, Merrifield developed the synthesis on

solid support[4] and could thereby overcome disadvantages as time-consuming

reactions and unforeseeable solubility characteristics of the intermediates. Nowadays,

solid phase peptide synthesis (SPPS) represents the method of choice for the

synthesis of sequence-specific peptides and small proteins (Figure 1).

Figure 1: Simplified representation of the workflow of solid phase peptide synthesis.

Briefly, the first amino acids is linked covalently to the resin, which enables the easy

removal of soluble unreacted reagents by washing and filtration of the resin and makes

the coupled peptide insoluble. By repeating the cycle of amino acid couplings and

deprotection, the peptide is synthesized step by step.[5,6] An excess of soluble reagents


4

during all steps should ensure an almost complete coupling and high yields. The

simplicity of the repetitive coupling of a new amino acid to the existing peptide and its

high reproducibility allows the automation of the entire process.[2]

In the most common SPPS strategies either an acid-labile (Boc) or a base-labile

(Fmoc) temporary protecting group is used. Both strategies require additional side

chain protecting groups that are stable to the conditions used for the removal of the

temporary, N-terminal protecting groups. However, while the Fmoc/tBu-strategy is

based on orthogonal protecting groups, the Boc/Bn-strategy is dependent on the

different stages of acid labilities.[7,8] During the lab course, only the Fmoc/tBu-strategy

will be used for peptide synthesis. Thus, the final deprotection of all protecting groups

and the cleavage of the peptide from the resin will be obtained by trifluoroacetic acid

(TFA). Peptides can then be analyzed regarding their identity (mass spectrometry) and

purity (HPLC) by different analytical procedures and purified by preparative reversed-

phase HPLC.


5

Block I – Resins and Linkers

In SPPS various solid phases (so-called resins) can be used. Most consist of

polystyrene that is cross-linked with divinylbenzene (1%). This matrix is relatively

inexpensive to produce and suitable for use in many common solvents

(dichloromethane (DCM), N,N'-dimethylformamide (DMF), N-methyl-pyrrolidone

(NMP)). An anchoring group (linker) facilitates the reversible connection between the

synthetic peptide chain and the solid phase. Additionally, it serves as a protecting

group for the C-terminal carboxyl group during chain elongation. Next-generation

resins contain PEG-based linker, which enables the synthesis of longer peptides and

complicated couplings on the resin. The choice of the linker determines the C-terminal

functional group of the final product. Most were designed to release free acids or acid

amides after cleavage.

The first step in solid phase peptide synthesis is the attachment or loading of the first

amino acid the linker of the resin. Since the yield of this reaction influences the yield of

the final product, this step should be as quantitative as possible. Unreacted groups (of

the linker) must acetylated (capped) by the reaction with acetic anhydride. This end-

capping prevents the formation of failure sequences during the subsequent coupling

cycles.


6

Loading of Rink amide-/ TG R RAM-Resin with the first Amino Acid

O

ONH

OMeO

OMe

Fmoc-Rink amide-Resin

Reagents

15 µmol Fmoc-Rink amide/ TG R

RAM-Resin20% piperidine in DMF

5 eq. Fmoc-amino acid

5 eq. hydroxybenzotriazole (HOBt)

(MW = 153.13 g/mol)

5 eq. diisopropylcarbodiimide (DIC)

(MW = 126.2 g/mol, = 0.806 g/ml)

DMF

Protocol

swelling the resin in 1 ml DMF for min. 15 min

deprotection of the Fmoc-protecting group: 2x incubation of the resin with 500 µl

20% piperidine in DMF for 15 min (RT, shaking)

washing of the resin: 5x DMF, 5x DCM, 5xDMF

coupling of the first amino acid: dissolve HOBt and Fmoc-amino acid in 300-500

µL DMF (depending on the resin used) (1.5 ml Eppi), add DIC

add reaction solution to the resin

reaction overnight (RT, shaking)

washing (5x DMF, 5x DCM)

dry in Speedvac if necessary


7

Loading of Wang-/TGR-HMP-Resin with the first Amino Acid

TGR-HMP-Resin

Reagents

15 µmol TGR-HMP-Resin



(MW = 153.13 g/mol)


(MW = 126.2 g/mol, = 0.806 g/ml)

DMF

Protocol


coupling of the first amino acid: dissolve HOBt and Fmoc-amino acid in 300-500

µl DMF (depending on the resin used) (1.5 ml Eppi), add DIC


reaction over night (RT, shaking)




8

Loading of Cl-Trt-Resin with the first Amino Acid

Protocol

swelling the resin in 1 ml dry DCM for 15 min

coupling of the first amino acid: dissolve Fmoc-amino acid in 600 µl DCM (1.5

ml Eppi), add DIPEA


reaction overnight (RT, shaking)



Chemicals

30 mg Chlorotrityl-Resin


12 eq. Diisopropylethylamine(DIPEA)

(MW 129,25 g/mol, 0,755 g/ml)

DCM

To enable the cleavage of a fully

protected peptide, chlorotrityl resin can

be used. After cleavage, a free acid will

be obtained.

Cl

Cl

Chlorotrityl-Harz


9

Determination of resin loading

To determine the loading of the resin, the

Fmoc-group of a defined amount of resin

is cleaved. The amount of the resulting

fluorenyl group in the supernatant is

subsequently measured by UV-Vis

spectroscopy at 300 nm.

Reagents

approx. 2-3 mg resin

20% piperidine in DMF

DMF

Protocol

weigh in about 2-3 mg dry resin in a 15 ml falcon tube and write down the

exact amount !

add exactly 0.5 mL 20% piperdine in DMF to the resin beads and centrifuge

let it react for 30 min at RT on a shaker and subsequently centrifuge

add 3 mL DMF and vortex, additionally prepare 1:10 dilution

prepare blank solution

the absorption of the blank and the sample is measured at 300 nm (for each

measurement 2 mL solution in a cuvette)

Calculation of the resin loading

The resin loading L300 in mmol/g is calculated by using the Lambert-Beer equation:

dcE

E = extinction; ε = extinction coefficient (7800 l mol-1 cm-1)

Derive the equation for determining the resin loading (L300).

(This equation is only valid in the linear range up to an E= 0.8. If a higher E is

measured: adjust the dilution!)

Pay Attention: If no satisfying loading is obtained, a repetition of the coupling

is needed!

http://dict.leo.org/#/search=cuvette&searchLoc=0&resultOrder=basic&multiwordShowSingle=on

http://dict.leo.org/#/search=extinction&searchLoc=0&resultOrder=basic&multiwordShowSingle=on

http://dict.leo.org/#/search=coefficient&searchLoc=0&resultOrder=basic&multiwordShowSingle=on


10

Endcapping

Endcapping means the blocking of

free binding sites on the resin

surface.

For Rink amide resins, acetylation of

free amino groups occurs with acetic

anhydride.

Reagents for Rink amide/TG R

RAM/Wang resin

50 µl acetic anhydride (Ac2O)

(MW = 102.09 g/mol, = 1.082 g/ml)

50 µl diisopropylethylamine (DIPEA)

(MW = 129.25 g/mol, = 0.755 g/ml)

DCM

Protocol

swelling the resin in 1 ml DCM for 15 min

dissolve acetic anhydride and DIPEA in 400 µl of DCM, add to resin

reaction for 15 min (RT, shaking)

washing (5x DMF, 5x DCM) and dry in Speedvac if necessary

Cl-Trt resins can be easily

capped with methanol.

Reagents for Cl-Trt resin

50 µl dry MeOH


(MW = 129.25 g/mol, = 0.755 g/ml)

DCM

Protocol


mix MeOH with DIPEA in 350 µl of DCM and add to resin

reaction for 10 min (RT, shaking), twice


Bioorganic-Lab Solid Phase Peptide Synthesis

11

Block II – Coupling of Amino Acids

For the coupling of further amino acids, the C-terminus of amino acids must be

activated. Additionally, to avoid side reactions, the N-terminus and the side chains of

this amino acid must be protected. The coupling can be performed by a variety of

activating reagents, wherein the carboxyl carbon atom is activated by electron-

withdrawing groups to enable the electron-rich nucleophilic attack of the amino group.

A commonly used activator is N,N'-diisopropylcarbodiimide (DIC). It is characterized

by its good solubility in DMF. This is also the case for N,N'- diisopropylurea, which is

formed during activation and guaranteed the quantitative separation of DIC and the

urea derivative from the immobilized peptide at the end of each coupling step. In

general, the coupling is carried out with the addition of an epimerization suppressant,

e.g. Oxyma or hydroxybenzotriazole (HOBt). Thereby, during reaction, an active ester

is formed that shows no tendency for racemization. The coupling methods presented

here are just a few of many varieties. A large number of other reagents are available

for standard reaction but also for difficult, hindered couplings.


12

Course of Standard Coupling of Amino Acids

step to do reagents times time [min]

1 swelling DMF 1 15

2 Fmoc-deprotection 20% piperidine in DMF 1 15

3 Fmoc-deprotection 20% piperidine in DMF 1 15

4 washing 5x DMF, 5x DCM, 5x DMF

5* coupling Fmoc-AA/HOBt/DIC 1 60-180

6* washing 5x DMF, 5x DCM, 5x DMF

7** washing/drying DCM

* In a double coupling, step 5 and 6 are repeated.

** Resins are dried in vacuo (SpeedVac) and stored at 4°C if you do not continue with

any reactions


13

Fmoc-Deprotection

Fmoc-protecting group

Reagents

20% piperidine in DMF

DMF

Protocol

swelling the resin in 1 ml DMF for 15 min

add 500 µL of a piperidine solution to the resin


alternative: (for existing ester linkages or complex structures!)

Fmoc-deprotection using DBU/piperidine

swelling the resin in 1 ml DMF for 15 min

2 x 1 ml DBU/piperidine in DMF (1:1:8) for 20 sec each

http://upload.wikimedia.org/wikipedia/commons/a/ae/Fmoc-NR.svg


14

Manual amino acid coupling

1. Oxyma/DIC

Oxyma

Reagents


5 eq. ethylcyanoglyoxylat-2-oxim (Oxyma)

(MW = 142.11 g/mol)


(MW = 126.2 g/mol, = 0.806 g/ml)

DMF

Protocol


weigh out Fmoc-amino acid and Oxyma in separate 1.5 ml Eppis

dissolve Fmoc-amino acid and Oxyma each in 150-250 µl DMF (depending on

resin used)

add Oxyma to the resin and pre-incubate for 2 min

add dissolved amino acid and DIC

reaction for 1-3 h, or overnight (RT, shaking)

for double coupling:

o washing (5x DMF, 5x DCM, 5x DMF)

o reaction 1-3 h (RT, shaking)


dry on vacuum pump for Kaisertest or Speedvac for cleavage from resin


15

2. HOBt/DIC

N

N

N

OH

N

CH3

CH3

N

CH3

CH3

HOBt DIC

Reagents



(MW = 153.13 g/mol)


(MW = 126.2 g/mol, = 0.806

g/ml)

DMF

Protocol


dissolve HOBt und Fmoc-amino acid in 300-500 µl DMF (depending on the

resin used) , add DIC


reaction 3h or overnight (RT, shaking)

for double coupling:

o washing (5x DMF, 5x DCM, 5x DMF)

o reaction 3h (RT, shaking)


dry on vacuum pump for Kaisertest or Speedvac for cleavage from resin


16

Kaisertest

Free amino groups can be detected by the Kaisertest offering the possibility to check

the completeness of couplings.

Reagents

solution I : 1.0 g ninhydrine in 20 ml EtOH

solution II : 80 g phenole in 20 ml EtOH

solution III : 0.4 ml aqueous KCN-solution (1mM) in 20 ml pyridine

(solutions are already prepared, ask supervisor)

Protocol

put some resin-beads (use only dry resin) in a 1.5 ml Eppi and add 1 drop of

solution I,II,III, respectively

a vessel without resin serves as control

the reaction solution is heated to 95°C in the thermomixer without shaking for 5

min

phenole: KCN: SEPERATE WASTE!

colored yellow : none free amino groups

colored blue: free amino groups (incomplete coupling)


17

Block III – Synthesis / Elongation of a Peptide in a

Synthesis Robot

In principle, solid phase peptide synthesis is carried out nowadays fully automated by

a synthesis robot. The device is a specially equipped pipetting robot, which is controlled

by PC software. Up to 96 different peptides (depending on model) can be synthesized

simultaneously. The synthesis runs in polypropylene syringes that are equipped with

frit plates. These syringes are attached to a special holder, the so-called extraction

block and are filled using pipetting arms. In principle, the synthesis is carried out as

following:

After filling the syringes with the desired resins, the sequences of the peptides and the

synthesis information are entered to the control computer. The computer calculates

the amounts of all chemicals that are required for the synthesis (DMF, Fmoc-AA in

Oxyma/DMF, piperidine in DMF, DIC in DMF). Subsequently, these reagents are filled

into the appropriate storage containers and then the synthesis is started. To extend

the peptide chain by one amino acid, the robot performs the following steps:

I. Removal of the Fmoc protecting group of the amino acid located at the resin by

piperidine / DMF

II. Washing of the syringes with DMF

III. Coupling of the following amino acid by activation with DIC / Oxyma

IV. Washing of the syringes with DMF

Abb. 3: Synthesis robot of MultiSyntech.


18

In detail, the following reagents and settings are used:

Company: MultiSyntech

Type: Syro 1

Resin: 15 µmol

AA: 8eq. (120 µmol) 0.3 M dissolved in 0.1 M HOBt/DMF

DIC: 8eq. (120 µmol) 1.32 M in DMF

Oxyma: 8eq. (120 µmol) 1.2 M in DMF

Piperidine: 40% solution in DMF

System liquid: DMF

1st cycle: swelling for 10 min in DMF, 800 µL

Fmoc-deprotection 3 min 40% piperidine, 600 µL

Fmoc-deprotection 10 min 20% piperidine, 300 µL + 300 µL

4 wash cycles with DMF, 600 µL

AA coupling for 42 min (2 min Oxyma and AA on resin,

then DIC is added), 400 µL + 100 µL + 100 µL

1 wash cycle with DMF, 600 µL


then DIC is added) 400 µL + 100 µL + 100 µL

2 wash cycles, 600 µL




2nd – nth cycle: AA coupling for 42 min (2 min Oxyma and AA on resin,


1 wash cycle with DMF



2 wash cycles, 600 µL





19

Block IV - Peptide Analytics

Owing to the fact that a peptide cannot be analyzed during the synthesis process

directly on the resin, a sample splitting is performed first. The cleavage of the peptide

from the resin and the removal of all side chain protecting groups occurs by

trifluoroacetic acid with the addition of scavengers (radical scavenger). During the

cleavage, highly reactive cationic species are formed from the protecting groups and

resin linkers. If the cations are not intercepted, they can react with amino acids that

have electron-rich functional groups (tyrosine, tryptophan, methionine, cysteine).

Electron-rich aromatics such as thioanisole or thiocresol are mainly applied as

scavengers, but also triisopropylsilane (TIS) or the reducing 1,2-ethanedithiol (EDT)

are used. After the peptide was precipitated in diethyl ether, it is analyzed by analytical

reversed-phase HPLC and MALDI-TOF mass spectrometry. RP-HPLC is used to

identify the purity of the compounds and is based on the different affinities of peptide

and by-products to the stationary phase. Generally, C18- and C12-based columns are

used in peptide chemistry as stationary phases and H2O and ACN with TFA serve as

mobile phase. To determine the identity of the samples, mass spectrometry is carried

out. Most applicable for peptides are MALDI-TOF and ESI-ion trap or -quadrupole

mass spectrometry.

For analysis in the lab course, you have to give the following information:

HPLC: your name, peptide name, concentration, method, expected elution (ACN

concentration)

MS: your name, peptide name, concentration, monoisotopic mass, average mass


20

Test Cleavage

Complete coupling can be checked by test

cleavages. For analytical purposes (HPLC,

MS) a small amount of peptide is cleaved

from the resin.

Reagents

90 µl trifluoroacetic acid (TFA)

(Gloves! Safety glasses!)

10 µl scavenger

dry ether

Scavenger (please ask supervisor before)

for peptides without Cys, Trp, Met Thiokresol/Thioanisol (1:1)

or TIS/water

for peptides with Cys, Trp, Met Thioanisol/Ethandithiol (7:3)

or alternative

Protocol

put some resin-beads in a 1.5 ml Eppi, add scavenger and subsequently TFA

reaction 2 h (RT, shaking), if peptide is sensitive to light, cover the tube in

aluminum foil

to precipitate the cleaved peptide, 1 ml of ice-cold ether is added and the

solution is kept for at least 20 min in the freezer

the precipitate is centrifuged and washed until no scavenger smell can be

observed (min. 5 times)

dry in Speedvac

dissolve the precipitate in 50 - 150 µl H2O/ACN, depending on peptide amount,

and separate the resin by centrifugation

Analytics:

MS: 10 µl peptide solution

HPLC: 10-20 µl peptide solution + 40-30 µl acetonitrile /H2O

(always ask supervisor)


21

Full Cleavage

After the successful synthesis, the entire peptide

is cleaved from the resin by a full cleavage.

During this process, the protection groups that

were required for the synthesis, are also

cleaved.

Reagents

900 µl trifluoroacetic acid

(TFA)

(Gloves! Safety glasses!)

100 µl scavenger

dry ether

Scavenger (ask supervisor before)

for peptides without Cys, Trp, Met Thiokresol/Thioanisol (1:1)

or TIS/water

for peptides with Cys, Trp, Met Thioanisol/Ethandithiol (7:3)

or alternative

Protocol

weigh out a new 15 ml tube without the lid, write down exact mass!

add scavanger to the resin and subsequently TFA and shake for 3h at RT

if peptide is sensitive to light, cover the tube in aluminum foil

to precipitate the cleaved peptide, the reaction solution is transferred in the

weighed 15 ml tube filled with 10 ml of ice-cold ether, for a complete precipitation

the tube is kept for at least 20 min in the freezer

the precipitate is centrifuged and washed until no smell can be observed

dry in Speedvac

dissolve the precipitate in 1 - 3 ml H2O/ACN, depending on peptide amount

analytics: (always ask supervisor)


HPLC: 10-20 µl peptide solution + 40-30 µl acetonitrile/H2O

The peptide solution is to be lyophilized.


22

Cleavage of fully protected peptides

The Cl-trityl resin is used to cleave fully protected

peptide fragments from solid support and using

them for further modification.

Reagents

DCM

TFE

glacial acetic acid

Protocol

swelling of the resin in 1 h in DCM

add glacial acetic acid/TFE/DCM (1:1:8) to the resin and shake it over night at

4°C

repeat with new solution for 3h at RT

transfer the reaction solution into a yellow cap and wash at least 10 times with

DCM until the acetic acid smell is gone

evaporate solvents

dissolve the pellet in 0,5-1 ml DCM, transfer the solution into a new tube and

evaporate the solution

the precipitate is dissolved in 1-3 ml H2O/ACN

analytics:




The peptide solution is transferred into a tared Falcon tube and lyophilized.

http://www.dict.cc/englisch-deutsch/glacial.html

http://www.dict.cc/englisch-deutsch/acetic.html

http://www.dict.cc/englisch-deutsch/acid.html

http://www.dict.cc/englisch-deutsch/glacial.html

http://www.dict.cc/englisch-deutsch/acetic.html

http://www.dict.cc/englisch-deutsch/acid.html


23

Methionine reduction

Protocol

Solve dried peptide in 1 ml TFA

add 16 µl EDT and 12 µl TMSBr

shake for 30 min at RT

precipitate in 20 ml ice-cold ether and incubate it in the freezer for at least 20

min

the precipitate is centrifuged and washed until no scavenger smell can be

observed (min. 5 times)

dry in Speedvac

dissolve the precipitate in 1 - 3 ml H2O/ACN, depending on peptide amount

analytics:




The peptide solution is to be lyophilized.

Reagents

1 mL TFA

16 μL Ethandithiole (EDT)

12 μL Trimethylsilylbromide (TMSBr)

Ice-cold ether

Twice oxidized methionine


24

Block V – Peptide Modifications on Solid Support

Modifications of peptides are of great interest due to their application. Functional

groups or molecules as fluorophores, radioactive groups or biotin serve as a reporter

group. On the other hand, biologically active agents (cytotoxic agents) or polyethylene

glycol (PEG) chains are interesting for the function and the metabolic stability of the

peptide. These are just a few examples, the range of modifications is very broad and,

accordingly, there are also different synthesis strategies. In most cases, it is useful to

couple the appropriate group already on the solid phase, as this reduces side reactions

that may occur at a synthesis in solution.


25

Dde Cleavage

The Dde protecting

group can be cleaved from

the resin using 2%

hydrazine.

Conditions during Fmoc

and Boc cleavage do not

induce Dde removal.

Dde

O O

NH

Peptide

Reagents

Freshly prepared!

15 ml Cleavage solution:

2% hydrazine (v/v)

in DMF

In case you have an

allyl/alloc group on

your peptide:

200 eq. allyl alcohol

Protocol

swelling of the resin in 1 ml DMF for 15 min

incubate resin with 1 ml hydrazine solution

react for 10 min (RT, shake), wash twice with DMF

repeat at least 10 times

increase to 3% hydrazine, if cleavage is not complete after 15 times

wash thoroughly with DMF or DMF/DCM

UV-Absorption: testing completeness of Dde-cleavage

collect the flow-through of the first cleavage reaction and the two washing

steps (1 ml DMF)

do the same for the last reaction

prepare blank

measure the absorption at 301 nm (2 ml)!

the cleavage is complete if the absorption of the 10th fraction is < 0.1.


26

Trt protection

Protocol

swelling of the resin in 1 ml DCM for 15 min

dissolve Trityl-Cl in DCM, add DIPEA

add to resin, incubate over night

wash protocol: 10x DCM, 5x DMF

Reagents

10 eq. Trityl-Cl

20 eq. DIPEA

1 ml DCM


27

Boc protection

Protocol


dissolve di-tert-butyl-dicarbonate in DCM, add DIPEA

add to resin, incubate 2h

wash 5x DCM, 5x DMF, 5x DCM, dry

Kaisertest

Reagents

10 eq. Di-tert-butyl-dicarbonate (MW = 218.25 Da)

1 eq. DIPEA (MW = 129.25 Da, ρ= 0.755 g/ml)

500 µl DCM


28

Mtt/Mmt/Trt/Opp Cleavage

Protocol


incubate resin with 15 x 1 mL of TFA solution (shake before adding)

reaction time 60 s (RT, shaking) and afterwards washing with DCM

wash protocol: 10x DCM, 5x DMF

If possible, always use Dde as the orthogonal

protecting group instead of Mtt!!!

Reagents

15 mL DCM:TIS:TFA (94:5:1)

Mtt protecting groups at lysine and Trt

protecting groups at serine can be cleaved by

incubation with 1% TFA in DCM in the

presence of 5% scavenger (TIS). The

protecting group is acid labile and will be

stable during Fmoc cleavage.


29

Cyclization of fully protected peptide

Cyclisation of a peptide can be

carried out on a resin. Therefore, two

reactive groups have to be

selectively deprotected and coupled

with HOBt and DIC.

Reagents


(MW = 153.13 g/mol)


(MW = 126.2 g/mol, = 0.806 g/ml)

14 mL DCM

Protocol

dissolve HOBt in 100 µL DMF

dissolve DIC and fully protected peptide in 14 mL DCM and add HOBT to this

mixture

Incubation: overnight (RT, shaking)

Evaporate DCM

Wash and evaporate with 1 mL DCM 5 times

dry in vacuo

Kaisertest or analytics


30

General coupling of carboxylic acids to amines

N

N

N

OH

N

CH3

CH3

N

CH3

CH3

HOBt DIC

Reagents

5 eq. reagent carrying carboxylic

acid


(MW = 153.13 g/mol)


(MW = 126.2 g/mol, = 0.806

g/ml)

250 µl DMF

Protocol

Swell resin in 1 mL DMF for at least 15 min

Dissolve carboxylic acid carrying reagent and HOBt in 300-500 µl DMF

(depending on the resin used), add DIC and give this mixture to swollen resin

Incubation: 3 h (RT, shaking)


31

Coupling of special amino acids

N

N

N

OH

N

CH3

CH3

N

CH3

CH3

HOBt DIC

Reagents

1 - 3 eq. amino acid


(MW = 153.13 g/mol)


(MW = 126.2 g/mol, = 0.806

g/ml)

250 µl DMF

Protocol


Dissolve carboxylic acid carrying reagent and HOBt in 300-500 µl DMF

(depending on the resin used), add DIC and give this mixture to swollen resin

Incubation: over night (RT, shaking)

If you use unnatural amino acids, you most likely have to use

less equivalents due to price and availability.

Always ask supervisor!


32

TAMRA-coupling

TAMRA is a fluorescence dye and

needs to be coupled at the end of the

synthesis, as it is sensitive to basic

treatment.

Reagents

15 µmol resin

2 eq. 6-Carboxytetramethylrhodamine

(MW = 430.45 g/mol)

1.9 eq. HATU (MW = 380.23 g/mol)

2 eq. DIPEA (MW = 129.25 g/mol, ρ =

0.755 g/mL)

DMF

Protocol

Swell resin in 1 mL DMF for at least 15 min.

Dissolve TAMRA and HATU in 500 µL DMF and add this mixture to swollen

resin. Add DIPEA directly into synthesis syringe.

Incubation: 2 h or overnight in the dark (RT, shaking)

Washing (5 x DMF, 5 x DCM)

Dry in vacuo


Fluorescent dye has to be protected from light!


33

CF-coupling

CF is a fluorescence dye and needs

to be coupled at the end of the

synthesis, as it is sensitive to basic

treatment.

Reagents

15 µmol resin

2 eq. 5-Carboxyfluorescein

(MW = 376.32 g/mol)

1.9 eq. HATU (MW = 380.23 g/mol)

2 eq. DIPEA (MW = 129.25 g/mol, ρ =

0.755 g/mL)

DMF

Protocol

Swell resin in 1 mL DMF for at least 15 min.

Dissolve CF and HATU in 500 µL DMF and add this mixture to swollen resin.

Add DIPEA directly into synthesis syringe.

Incubation: 2 h or overnight in the dark (RT, shaking)

Washing (10 x DMF)

Polymer cleavage: Add 20% piperidine in DMF to swollen resin and incubate for

45 min (RT, shaking).


Dry in vacuo


Fluorescent dye has to be protected from light!


34

Coupling of octanoic acid

Octanoic acid is coupled to a

hydroxyl group to from an ester bond.

Reagents

5 eq. octanoic acid

5 eq. DIC

5 eq. HOBt

5 eq. methylimidazole

0.1 eq. DMAP

DMF

Protocol


preactivate with DIC in 150-250 µl DMF (depending on resin used) for 10 min

Dissolve octanoic acid, HOBt, MEI and DMAP in in 150-250 µl DMF (depending

on resin used)and add to resin

Incubation: overnight in the dark (RT, shaking)


Dry in vacuo



35

Biotin Labeling

S

NN

O

COOH

H H

(+)-Biotin

Reagents

3 eq. (+)-Biotin

(MW = 244.3 g/mol)

3 eq. Hydroxybenzotriazole (HOBt)

(MW = 153.13 g/mol)

3 eq. Diisopropylcarbodiimide (DIC)

(MW = 126.2 g/mol, = 0.806

g/ml)

NMP

Protocol

swelling of the resin in 1 ml DMF for 15 min

dissolve biotin and HOBt in 600 µl NMP incubate tube at 50°C until

Biotin/HOBt is completely dissolved

add DIC and transfer solution to the resin

reaction over night (RT, Shaking)


dry in vacuo



36

Acetylation

Acetylation serves to protect the N-

terminus of a peptide against

cleavage or in order to block the N-

terminal charge in a peptide

sequence taken out of a protein.

Reagents

50 µl acetanhydride (Ac2O)

(MW = 102.09 g/mol, = 1.082 g/ml)


(MW = 129.25 g/mol, = 0.755 g/ml)

DCM

Protocol


dissolve acetanhydride and DIPEA in 400 µl of DCM, add to resin




37

Benzylamin

Reagents

50 eq. benzylamin

(MW = 107.15 g/mol)

50 eq. Hydroxybenzotriazol (HOBt)

(MW = 153.13 g/mol)

50 eq. Diisopropylcarbodiimid (DIC)

(MW = 126.2 g/mol, = 0.806 g/ml)

50 eq. DIPEA

(MW = 129.25 g/mol; = 0.755 g/mL)

DMF/DCM

Protocol

synthesize peptide on Chlorotrityl resin

remove from resin fully protected

dissolve in DCM/DMF

add DIC, HOBt, DIPEA

shake over night at room temperature

liquid-liquid extraction (keep both phases)

o acidic extraction (twice 5 ml 0.1 M NH4Cl)

o basic extraction (twice 5 ml 0.1 M NH4CO3)

dry organic phase and dissolve peptide in ACN/H2O/tBuOH

o MS and HPLC

lyophilize peptide and add 1 ml TFA/TA/TK (90:5:5), shake 3h

precipitate with 10 ml diethyl ether, wash

RP-HPLC and MS


38

Methylene thioacetal formation

Reagents

3 M aqueous K2CO3

1.5 eq. TCEP

12 eq. triethylamine

8 eq. diiodomethane

THF

Protocol

purified peptide dissolved in 3 M aqueous K2CO3

add TCEP and stir for 1h

dissolve triethylamine and diiodomethane separately in THF

add stepwise alternating triethylamine and diiodomethane

stir for 12h


39

References

[1] Ahrens, Bellmann-Sickert, Beck-Sickinger: Peptides and Peptide Conjugates:

Therapeutics on the Upward Path, 2012, Future Med Chem, 4, 1567.

[2] Mäde, Els-Heindl, Beck-Sickinger: Automated solid-phase peptide synthesis to

obtain therapeutic peptides, 2014, Beilstein J Org Chem, 10, 1197.

[3] Fischer, Fourneau: Ueber einige Derivate des Glykocolls, 1901, Ber Chem Ges, 34,

2868.

[4] Merrifield: Solid Phase Peptide Synthesis I: The Synthesis of a Tetrapeptide, 1963,

JACS, 85, 2149.

[5] Montalbetti, Falque: Amide bond formation and peptide coupling, 2005,

Tetrahedron 61, 10827.

[6] Altmann, Mutter: Die chemische Synthese von Peptiden und Proteinen, 1993,

Chemie in unserer Zeit, 6, 274.

[7] https://www.bachem.com/fileadmin/user_upload/pdf/Catalogs_Brochures/Solid

_Phase_Peptide_Synthesis.pdf

[8] Isidro-Llobet, Alvarez, Albericio: Amino acid-protecting groups, 2009, Chem Rev,

109, 2455.


40

GHS hazard statement

TFA:

GHS 05 – Ätzend 07 – Achtung Gefahr

H: 332‐314‐412

P: 271‐273‐301+330+331‐305+351+338‐309+310 [1]

DCM:

GHS 08 – Gesundheitsgefährdend 07 – Achtung

H: 315‐319‐335‐336‐351‐373

P: 261‐281‐305+351+338

DMF:

GHS 02 – Leicht-/Hochentzündlich 07 – Achtung 08 – Gesundheitsgefährdend

H: 360D‐226‐332‐312‐319

P: 201‐302+352‐305+351+338‐308+313

DIC:

GHS 02 – Leicht-/Hochentzündlich 05 – Ätzend 06 – Giftig oder sehr giftig 08 –

Gesundheitsgefährdend

H: 226‐315‐317‐318‐330‐334‐335

P: 260‐280‐284‐305+351+338‐310

ACN:

GHS 02 – Leicht-/Hochentzündlich 07 – Achtung

H: 225‐332‐302‐312‐319

P: 210‐305+351+338‐403+235

English Version see from page 53


41

GHS01 Explosivstoffe

können durch Schlag, Reibung, Feuer oder andere Zündquellen auch ohne

Beteiligung von Luftsauerstoff explodieren.

GHS02 Entzündlich

a) Flüssigkeiten mit Flammpunkt < 0°C und Siedpunkt ~ 35°C b) Gase, die unter Normalbedingungen bei Luftkontakt entzündlich sind c) Feststoffe, die bei kurzzeitiger Zündquellen-Einwirkung leicht entzündet

werden d) Flüssigkeiten mit Flammpunkt < 21°C e) Stoffe, die bei gewöhnlicher Temperatur sich an Luft erhitzen und entzünden f) Stoffe, die bei Feuchtigkeit hochentzündliche Gase in gefährlicher Menge

entwickeln.

GHS03 Brandfördernde Stoffe und org. Peroxide

i. d. R. selbst nicht brennbar, erhöhen aber bei Berührung mit brennbaren Stoffen

(durch Sauerstoffabgabe) die Brandgefahr und Brandheftigkeit.

GHS04 Gase unter Druck

GHS05 Korrosive Stoffe

können bei Berührung lebendes Gewebe zerstören (z.B. Säuren mit

pH ~ 2, Laugen mit pH ~ 11,5),

GHS06 Giftige Stoffe

können in sehr geringer Menge beim Einatmen, Verschlucken oder

Hautresorption zum Tode oder zu akuten oder chronischen Gesundheitsschäden

führen,


42

GHS07 Reizende oder sensibilisierende Stoffe (Haut, Augen)

a) können bei Kontakt deutliche Entzündungen der Haut oder Augenschäden hervorrufen.

b) können bei Hautkontakt Überempfindlichkeitsreaktionen (Allergien) auslösen.

GHS08 reizende oder sensibilisierende Stoffe (Einatmung), Krebserregende, mutagene,

teratogene Stoffe

a) können beim Menschen Krebs erregen oder die Krebshäufigkeit erhöhen b) können beim Menschen vererbbare genetische Schäden hervorrufen c) können beim Menschen die Fortpflanzungsfähigkeit beeinträchtigen oder

fruchtschädigend wirken

Kat. 1: bekanntermaßen

Kat. 2: angenommen aufgrund hinreichender Anhaltspunkte

Kat. 3: geben wegen möglicher krebserzeugender Wirkung beim Menschen

Anlass zu Besorgnis – aufgrund einiger, jedoch nicht Ausreichender

Anhaltspunkte

GHS09 Umweltgefährdende Stoffe

können Naturhaushalt, Wasser, Boden, Luft, Klima, Tiere, Pflanzen oder Mikro-

organismen derart verändern, dass dadurch Gefahren für die Umwelt entstehen.

GEFAHRENHINWEISE (H-Sätze)

(Das Kodifizierungssystem für GHS - Gefahrenhinweise wird noch im UN – Sach-

verständigenausschuss diskutiert; deshalb könnten Änderungen notwendig sein.)

H200-Reihe: Physikalische Gefahren

H200 Instabil, explosiv

H201 Explosiv, Gefahr der Massenexplosion.

H202 Explosiv; große Gefahr durch Splitter, Spreng- und Wurfstücke.

H203 Explosiv; Gefahr durch Feuer, Luftdruck oder Splitter, Spreng- und Wurfstücke.

H204 Gefahr durch Feuer oder Splitter, Spreng- und Wurfstücke.

H205 Gefahr der Massenexplosion bei Feuer.

H220 Extrem entzündbares Gas.


43

H221 Entzündbares Gas.

H222 Extrem entzündbares Aerosol.

H223 Entzündbares Aerosol.

H224 Flüssigkeit und Dampf extrem entzündbar.

H225 Flüssigkeit und Dampf leicht entzündbar.

H226 Flüssigkeit und Dampf entzündbar.

H228 Entzündbarer Feststoff.

H240 Erwärmung kann Explosion verursachen.

H241 Erwärmung kann Brand oder Explosion verursachen.

H242 Erwärmung kann Brand verursachen.

H250 Entzündet sich in Berührung mit Luft von selbst.

H251 Selbsterhitzungsfähig; kann in Brand geraten.

H252 In großen Mengen selbsterhitzungsfähig; kann in Brand geraten.

H260 In Berührung mit Wasser entstehen entzündbare Gase, die sich spontan entzünden

können.

H261 In Berührung mit Wasser entstehen entzündbare Gase.

H270 Kann Brand verursachen oder verstärken; Oxidationsmittel.

H271 Kann Brand oder Explosion verursachen; starkes Oxidationsmittel.

H272 Kann Brand verstärken; Oxidationsmittel.

H280 Enthält Gas unter Druck; kann bei Erwärmung explodieren.

H281 Enthält tiefgekühltes Gas; kann Kälteverbrennungen oder -Verletzungen

verursachen.

H290 Kann gegenüber Metallen korrosiv sein.

H300-Reihe: Gesundheitsgefahren

H300 Lebensgefahr bei Verschlucken.


44

H301 Giftig bei Verschlucken.

H302 Gesundheitsschädlich bei Verschlucken.

H304 Kann bei Verschlucken und Eindringen in die Atemwege tödlich sein.

H310 Lebensgefahr bei Hautkontakt.

H311 Giftig bei Hautkontakt.

H312 Gesundheitsschädlich bei Hautkontakt.

H314 Verursacht schwere Verätzungen der Haut und schwere Augenschäden.

H315 Verursacht Hautreizungen.

H317 Kann allergische Hautreaktionen verursachen.

H318 Verursacht schwere Augenschäden.

H319 Verursacht schwere Augenreizung.

H330 Lebensgefahr bei Einatmen.

H331 Giftig bei Einatmen.

H332 Gesundheitsschädlich bei Einatmen.

H334 Kann bei Einatmen Allergie, asthmaartige Symptome oder Atembeschwerden

verursachen.

H335 Kann die Atemwege reizen.

H336 Kann Schläfrigkeit und Benommenheit verursachen.

H340 Kann genetische Defekte verursachen (Expositionsweg angeben, sofern schlüssig

belegt ist, dass diese Gefahr bei keinem anderen Expositionsweg besteht).

H341 Kann vermutlich genetische Defekte verursachen (Expositionsweg angeben, sofern

schlüssig belegt ist, dass diese Gefahr bei keinem anderen Expositionsweg besteht).

H350 Kann Krebs erzeugen (Expositionsweg angeben, sofern schlüssig belegt ist, dass

diese Gefahr bei keinem anderen Expositionsweg besteht).

H350 i Kann bei Einatmen Krebs erzeugen.

H351 Kann vermutlich Krebs erzeugen (Expositionsweg angeben, sofern schlüssig belegt

ist, dass diese Gefahr bei keinem anderen Expositionsweg besteht).


45

H360 Kann die Fruchtbarkeit beeinträchtigen oder das Kind im Mutterleib schädigen

(konkrete Wirkung angeben, sofern bekannt) (Expositionsweg angeben, sofern

schlüssig belegt ist, dass die Gefahr bei keinem anderen Expositionsweg besteht).

H360 F Kann die Fruchtbarkeit beeinträchtigen.

H360 D Kann das Kind im Mutterleib schädigen.

H360 FD Kann die Fruchtbarkeit beeinträchtigen. Kann das Kind im Mutterleib schädigen.

H360 Fd Kann die Fruchtbarkeit beeinträchtigen. Kann vermutlich das Kind im Mutterleib

schädigen.

H360 Df Kann das Kind im Mutterleib schädigen. Kann vermutlich die Fruchtbarkeit

beeinträchtigen.

H361 Kann vermutlich die Fruchtbarkeit beeinträchtigen oder das Kind im Mutterleib

schädigen (konkrete Wirkung angeben, sofern bekannt) (Expositionsweg angeben,

sofern schlüssig belegt ist, dass die Gefahr bei keinem anderen Expositionsweg

besteht)

H361 f Kann vermutlich die Fruchtbarkeit beeinträchtigen.

H361 d Kann vermutlich das Kind im Mutterleib schädigen.

H361 fd Kann vermutlich die Fruchtbarkeit beeinträchtigen. Kann vermutlich das Kind im

Mutterleib schädigen.

H362 Kann Säuglinge über die Muttermilch schädigen.

H370 Schädigt die Organe (oder alle betroffenen Organe nennen, sofern bekannt)

(Expositionsweg angeben, sofern schlüssig belegt ist, dass diese Gefahr bei keinem

anderen Expositionsweg besteht).

H371 Kann die Organe schädigen (oder alle betroffenen Organe nennen, sofern bekannt)

(Expositionsweg angeben, sofern schlüssig belegt ist, dass diese Gefahr bei keinem

anderen Expositionsweg besteht).

H372 Schädigt die Organe (alle betroffenen Organe nennen) bei längerer oder

wiederholter Exposition (Expositionsweg angeben, wenn schlüssig belegt ist, dass

diese Gefahr bei keinem anderen Expositionsweg besteht).

H373 Kann die Organe schädigen (alle betroffenen Organe nennen, sofern bekannt) bei

längerer oder wiederholter Exposition (Expositionsweg angeben, wenn schlüssig

belegt ist, dass diese Gefahr bei keinem anderen Expositionsweg besteht).

H400-Reihe: Umweltgefahren


46

H400 Sehr giftig für Wasserorganismen.

H410 Sehr giftig für Wasserorganismen mit langfristiger Wirkung.

H411 Giftig für Wasserorganismen, mit langfristiger Wirkung.

H412 Schädlich für Wasserorganismen, mit langfristiger Wirkung.

H413 Kann für Wasserorganismen schädlich sein, mit langfristiger Wirkung.

Ergänzende Gefahrenmerkmale und Kennzeichnungselemente (EUH-Sätze)

EUH 001 In trockenem Zustand explosiv.

EUH 006 Mit und ohne Luft explosionsfähig.

EUH 014 Reagiert heftig mit Wasser.

EUH 018 Kann bei Verwendung explosionsfähige/ entzündbare Dampf/ Luft-Gemische bilden.

EUH 019 Kann explosionsfähige Peroxide bilden.

EUH 044 Explosionsgefahr bei Erhitzen unter Einschluss.

EUH 029 Entwickelt bei Berührung mit Wasser giftige Gase.

EUH 031 Entwickelt bei Berührung mit Säure giftige Gase.

EUH 032 Entwickelt bei Berührung mit Säure sehr giftige Gase.

EUH 066 Wiederholter Kontakt kann zu spröder oder rissiger Haut führen.

EUH 070 Giftig bei Berührung mit den Augen.

EUH 071 Wirkt ätzend auf die Atemwege.

EUH 059 Die Ozonschicht schädigend.

EUH 201 Enthält Blei. Nicht für den Anstrich von Gegenständen verwenden, die von Kindern

gekaut oder gelutscht werden könnten.

201 A Achtung! Enthält Blei.

EUH 202 Cyanacrylat. Gefahr. Klebt innerhalb von Sekunden Haut und Augenlider zusammen.

Darf nicht in die Hände von Kindern gelangen.

EUH 203 Enthält Chrom(VI). Kann allergische Reaktionen hervorrufen.


47

EUH 204 Enthält Isocyanate. Kann allergische Reaktionen hervorrufen.

EUH 205 Enthält epoxidhaltige Verbindungen. Kann allergische Reaktionen hervorrufen.

EUH 206 Achtung! Nicht zusammen mit anderen Produkten verwenden, da gefährliche Gase

(Chlor) freigesetzt werden können.

EUH 207 Achtung! Enthält Cadmium. Bei der Verwendung entstehen gefährliche Dämpfe.

Hinweise des Herstellers beachten. Sicherheitsanweisungen einhalten.

EUH 208 Enthält (Name des sensibilisierenden Stoffes). Kann allergische Reaktionen

hervorrufen.

EUH 209 Kann bei Verwendung leicht entzündbar werden.

209 A Kann bei Verwendung entzündbar werden.

EUH 210 Sicherheitsdatenblatt auf Anfrage erhältlich.

EUH 401 Zur Vermeidung von Risiken für Mensch und Umwelt die Gebrauchsanleitung

einhalten.

SICHERHEITSHINWEISE (P-Sätze)

P 100-Reihe: Allgemeines

P101 Ist ärztlicher Rat erforderlich, Verpackung oder Kennzeichnungsetikett bereithalten.

P102 Darf nicht in die Hände von Kindern gelangen.

P103 Vor Gebrauch Kennzeichnungsetikett lesen.

P 200-Reihe: Prävention

P201 Vor Gebrauch besondere Anweisungen einholen.

P202 Vor Gebrauch alle Sicherheitshinweise lesen und verstehen.

P210 Von Hitze / Funken / offener Flamme / heißen Oberflächen fernhalten nicht rauchen.

P211 Nicht gegen offene Flamme oder andere Zündquelle sprühen.

P220 Von Kleidung /…/ brennbaren Materialien fernhalten/entfernt aufbewahren.

P221 Mischen mit brennbaren Stoffen /… unbedingt verhindern.


48

P222 Kontakt mit Luft nicht zulassen.

P223 Kontakt mit Wasser wegen heftiger Reaktion und möglichem Aufflammen unbedingt

verhindern.

P230 Feucht halten mit …

P231 Unter inertem Gas handhaben.

P232 Vor Feuchtigkeit schützen.

P233 Behälter dicht verschlossen halten.

P234 Nur im Originalbehälter aufbewahren.

P235 Kühl halten.

P240 Behälter und zu befüllende Anlage erden.

P241 Explosionsgeschützte elektrische Betriebsmittel / Lüftungsanlagen / Beleuchtung /…

verwenden.

P242 Nur funkenfreies Werkzeug verwenden.

P243 Maßnahmen gegen elektrostatische Aufladungen treffen.

P244 Druckminderer frei von Fett und Öl halten.

P250 Nicht schleifen / stoßen /…/ reiben.

P251 Behälter steht unter Druck: Nicht durchstechen oder verbrennen, auch nicht nach der

Verwendung.

P260 Staub / Rauch / Gas / Nebel / Dampf / Aerosol nicht einatmen.

P261 Einatmen von Staub/Rauch/Gas/Nebel/Dampf/Aerosol vermeiden.

P262 Nicht in die Augen, auf die Haut oder auf die Kleidung gelangen lassen.

P263 Kontakt während der Schwangerschaft / und der Stillzeit vermeiden.

P264 Nach Gebrauch … gründlich waschen.

P270 Bei Gebrauch nicht essen, trinken oder rauchen.

P271 Nur im Freien oder in gut belüfteten Räumen verwenden.

P272 Kontaminierte Arbeitskleidung nicht außerhalb des Arbeitsplatzes tragen.

P273 Freisetzung in die Umwelt vermeiden.


49

P280 Schutzhandschuhe / Schutzkleidung / Augenschutz / Gesichtsschutz tragen.

P281 Vorgeschriebene persönliche Schutzausrüstung verwenden.

P282 Schutzhandschuhe / Gesichtsschild / Augenschutz mit Kälteisolierung tragen.

P283 Schwer entflammbare / flammhemmende Kleidung tragen.

P284 Atemschutz tragen.

P285 Bei unzureichender Belüftung Atemschutz tragen.

P231 +

P232

Unter inertem Gas handhaben. Vor Feuchtigkeit schützen.

P235 +

P410

Kühl halten. Vor Sonnenbestrahlung schützen.

P 300-Reihe: Reaktion

P301 BEI VERSCHLUCKEN:

P302 BEI BERÜHRUNG MIT DER HAUT:

P303 BEI BERÜHRUNG MIT DER HAUT (oder dem Haar):

P304 BEI EINATMEN:

P305 BEI KONTAKT MIT DEN AUGEN:

P306 BEI KONTAMINIERTER KLEIDUNG:

P307 BEI EXPOSITION:

P308 BEI EXPOSITION ODER FALLS BETROFFEN:

P309 BEI EXPOSITION ODER UNWOHLSEIN:

P310 Sofort GIFTINFORMATIONSZENTRUM oder Arzt anrufen.

P311 GIFTINFORMATIONSZENTRUM oder Arzt anrufen.

P312 Bei Unwohlsein GIFTINFORMATIONSZENTRUM oder Arzt anrufen.

P313 Ärztlichen Rat einholen / ärztliche Hilfe hinzuziehen.

P314 Bei Unwohlsein ärztlichen Rat einholen / ärztliche Hilfe hinzuziehen.


50

P315 Sofort ärztlichen Rat einholen / ärztliche Hilfe hinzuziehen.

P320 Besondere Behandlung dringend erforderlich (siehe … auf diesem

Kennzeichnungsetikett).

P321 Besondere Behandlung (siehe … auf diesem Kennzeichnungsetikett).

P322 Gezielte Maßnahmen (siehe … auf diesem Kennzeichnungsetikett).

P330 Mund ausspülen.

P331 KEIN Erbrechen herbeiführen.

P332 Bei Hautreizung:

P333 Bei Hautreizung oder -ausschlag:

P334 In kaltes Wasser tauchen / nassen Verband anlegen.

P335 Lose Partikel von der Haut abbürsten.

P336 Vereiste Bereiche mit lauwarmem Wasser auftauen. Betroffenen Bereich nicht

reiben.

P337 Bei anhaltender Augenreizung:

P338 Eventuell vorhandene Kontaktlinsen nach Möglichkeit entfernen weiter ausspülen.

P340 Die betroffene Person an die frische Luft bringen und in einer Position ruhigstellen,

die das Atmen erleichtert.

P341 Bei Atembeschwerden an die frische Luft bringen und in einer Position ruhigstellen,

die das Atmen erleichtert.

P342 Bei Symptomen der Atemwege:

P350 Behutsam mit viel Wasser und Seife waschen.

P351 Einige Minuten lang behutsam mit Wasser ausspülen.

P352 Mit viel Wasser und Seife waschen.

P353 Haut mit Wasser abwaschen / duschen.

P360 Kontaminierte Kleidung und Haut sofort mit viel Wasser abwaschen und danach

Kleidung ausziehen.

P361 Alle kontaminierten Kleidungsstücke sofort ausziehen.

P362 Kontaminierte Kleidung ausziehen und vor erneutem Tragen waschen.


51

P363 Kontaminierte Kleidung vor erneutem Tragen waschen.

P370 Bei Brand:

P371 Bei Großbrand und großen Mengen:

P372 Explosionsgefahr bei Brand.

P373 KEINE Brandbekämpfung, wenn das Feuer explosive Stoffe / Gemische /

Erzeugnisse erreicht.

P374 Brandbekämpfung mit üblichen Vorsichtsmaßnahmen aus angemessener

Entfernung.

P375 Wegen Explosionsgefahr Brand aus der Entfernung bekämpfen.

P376 Undichtigkeit beseitigen, wenn gefahrlos möglich.

P377 Brand von ausströmendem Gas: Nicht löschen, bis Undichtigkeit gefahrlos beseitigt

werden kann.

P378 … zum Löschen verwenden.

P380 Umgebung räumen.

P381 Alle Zündquellen entfernen, wenn gefahrlos möglich.

P390 Verschüttete Mengen aufnehmen, um Materialschäden zu vermeiden.

P391 Verschüttete Mengen aufnehmen.

P301 +

P310

BEI VERSCHLUCKEN: Sofort GIFTINFOMATIONSZENTRUM oder Arzt anrufen.

P301 +

P312

BEI VERSCHLUCKEN: Bei Unwohlsein GIFTINFORMATIONSZENTRUM oder Arzt

anrufen.

P301 +

P330 +

P331

BEI VERSCHLUCKEN: Mund ausspülen. KEIN Erbrechen herbeiführen.

P302 +

P334

BEI KONTAKT MIT DER HAUT: In kaltes Wasser tauchen / nassen Verband

anlegen.

P302 +

P350

BEI KONTAKT MIT DER HAUT: Behutsam mit viel Wasser und Seife waschen.

P302 +

P352

BEI KONTAKT MIT DER HAUT: Mit viel Wasser und Seife waschen.


52

P303 +

P361 +

P353

BEI KONTAKT MIT DER HAUT (oder dem Haar): Alle beschmutzten, getränkten

Kleidungsstücke sofort ausziehen. Haut mit Wasser abwaschen/duschen.

P304 +

P340

BEI EINATMEN: An die frische Luft bringen und in einer Position ruhigstellen, die das

Atmen erleichtert.

P304 +

P341

BEI EINATMEN: Bei Atembeschwerden an die frische Luft bringen und in einer

Position ruhigstellen, die das Atmen erleichtert.

P305 +

P351 +

P338

BEI KONTAKT MIT DEN AUGEN: Einige Minuten lang behutsam mit Wasser spülen.

Vorhandene Kontaktlinsen nach Möglichkeit entfernen. Weiter spülen.

P306 +

P360

BEI KONTAKT MIT DER KLEIDUNG: Kontaminierte Kleidung und Haut sofort mit viel

Wasser abwaschen und danach Kleidung ausziehen.

P307 +

P311

BEI EXPOSITION: GIFTINFORMATIONSZENTRUM oder Arzt anrufen.

P308 +

P313

BEI EXPOSITION ODER FALLS BETROFFEN: Ärztlichen Rat einholen / ärztliche

Hilfe hinzuziehen.

P309 +

P311

BEI EXPOSITION ODER UNWOHLSEIN: GIFTINFORMATIONSZENTRUM oder

Arzt anrufen.

P332 +

P313

Bei Hautreizung: Ärztlichen Rat einholen / ärztliche Hilfe hinzuziehen.

P333 +

P313

Bei Hautreizung oder -ausschlag: Ärztlichen Rat einholen / ärztliche Hilfe

hinzuziehen.

P335 +

P334

Lose Partikel von der Haut abbürsten. In kaltes Wasser tauchen / nassen Verband

anlegen.

P337 +

P313

Bei anhaltender Augenreizung: Ärztlichen Rat einholen / ärztliche Hilfe hinzuziehen.

P342 +

P311

Bei Symptomen der Atemwege: GIFTINFORMATIONSZENTRUM oder Arzt anrufen.

P370 +

P376

Bei Brand: Undichtigkeit beseitigen, wenn gefahrlos möglich.

P370 +

P378

Bei Brand: … zum Löschen verwenden.

P370 +

P380

Bei Brand: Umgebung räumen.


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P370 +

P380 +

P375

Bei Brand: Umgebung räumen. Wegen Explosionsgefahr Brand aus der Entfernung

bekämpfen.

P371 +

P380 +

P375

Bei Großbrand und großen Mengen: Umgebung räumen. Wegen Explosionsgefahr

Brand aus der Entfernung bekämpfen.

P 400-Reihe: Aufbewahrung

P401 … aufbewahren.

P402 An einem trockenen Ort aufbewahren.

P403 An einem gut belüfteten Ort aufbewahren.

P404 In einem geschlossenen Behälter aufbewahren.

P405 Unter Verschluss aufbewahren.

P406 In korrosionsbeständigem /… Behälter mit korrosionsbeständiger Auskleidung

aufbewahren.

P407 Luftspalt zwischen Stapeln / Paletten lassen.

P410 Vor Sonnenbestrahlung schützen.

P411 Bei Temperaturen von nicht mehr als … °C / … aufbewahren.

P412 Nicht Temperaturen von mehr als 50 °C aussetzen.

P413 Schüttgut in Mengen von mehr als … kg bei Temperaturen von nicht mehr als … °C

aufbewahren

P420 Von anderen Materialien entfernt aufbewahren.

P422 Inhalt in / unter … aufbewahren

P402 +

P404

In einem geschlossenen Behälter an einem trockenen Ort aufbewahren.

P403 +

P233

Behälter dicht verschlossen an einem gut belüfteten Ort aufbewahren.

P403 +

P235

Kühl an einem gut belüfteten Ort aufgewahren.


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P410 +

P403

Vor Sonnenbestrahlung geschützt an einem gut belüfteten Ort aufbewahren.

P410 +

P412

Vor Sonnenbestrahlung schützen und nicht Temperaturen von mehr als 50 °C

aussetzen.

P411 +

P235

Kühl und bei Temperaturen von nicht mehr als … °C aufbewahren

P 500-Reihe: Entsorgung

P501 Inhalt / Behälter … zuführen.

GHS01 Explosive

Unstable explosives

Explosives, divisions 1.1, 1.2, 1.3, 1.4, 1.5, 1.6

Self-reactive substances and mixtures, types A, B

Organic peroxides, types A, B

GHS02 Flammable

g) Flammable gases, category 1

h) Flammable aerosols, categories 1, 2

i) Flammable liquids, categories 1, 2, 3, 4

j) Flammable solids, categories 1, 2

k) Self-reactive substances and mixtures, types B, C, D, E, F

l) Pyrophoric liquids, category 1

m) Pyrophoric solids, category 1

n) Combustible solids, category 3

o) Combustible liquids, category 3

p) Self-heating substances and mixtures, categories 1, 2

q) Substances and mixtures, which in contact with water, emit flammable gases, categories 1, 2, 3

r) Organic peroxides, types B, C, D, E, F

GHS03 Oxidizing

Oxidizing gases, category 1

Oxidizing liquids, categories 1, 2, 3

Oxidizing solids, categories 1, 2, 3


55

GHS04 Compressed Gas

Compressed gases

Liquefied gases

Refrigerated liquefied gases

Dissolved gases

GHS05 Corrosive

Corrosive to metals, category 1

GHS06 Toxic

Acute toxicity (oral, dermal, inhalation), categories 1, 2, 3

GHS07 Harmful

Acute toxicity (oral, dermal, inhalation), category 4

Skin irritation, categories 2, 3

Eye irritation, category 2A

Skin sensitization, category 1

Specific target organ toxicity following single exposure, category 3

o Respiratory tract irritation

o Narcotic effects

Not used[3]

with the "skull and crossbones" pictogram

for skin or eye irritation if:

o the "corrosion" pictogram also appears

o the "health hazard" pictogram is used to indicate respiratory sensitization

GHS08 Health hazard

https://en.wikipedia.org/wiki/GHS_hazard_pictograms#cite_note-3


56

Respiratory sensitization, category 1

Germ cell mutagenicity, categories 1A, 1B, 2

Carcinogenicity, categories 1A, 1B, 2

Reproductive toxicity, categories 1A, 1B, 2

Specific target organ toxicity following single exposure, categories 1, 2

Specific target organ toxicity following repeated exposure, categories 1, 2

Aspiration hazard, categories 1, 2

GHS09 Environmental hazard

Acute hazards to the aquatic environment, category 1

Chronic hazards to the aquatic environment, categories 1, 2

Environmental toxicity, categories 1, 2

Hazard statements

H200: Physical hazards

H200 Unstable explosive

H201 Explosive; mass explosion hazard

H202 Explosive; severe projection hazard

H203 Explosive; fire, blast or projection hazard

H204 Fire or projection hazard

H205 May mass explode in fire

H206 Fire, blast or projection hazard: increased risk of explosion if desensitizing agent is

reduced

H207 Fire or projection hazard: increased risk of explosion if desensitizing agent is reduced

H208 Fire hazard: increased risk of explosion if desensitizing agent is reduced

H220 Extremely flammable gas

H221 Flammable gas

H222 Extremely flammable aerosol

H223 Flammable aerosol

https://en.wikipedia.org/wiki/File:GHS-pictogram-silhouette.svg

https://en.wikipedia.org/wiki/File:GHS-pictogram-pollu.svg


57

H224 Extremely flammable liquid and vapour

H225 Highly flammable liquid and vapour

H226 Flammable liquid and vapour

H227 Combustible liquid

H228 Flammable solid

H229 Pressurized container: may burst if heated

H230 May react explosively even in the absence of air

H231 May react explosively even in the absence of air at elevated pressure and/or temperature

H232 May ignite spontaneously if exposed to air

H240 Heating may cause an explosion

H241 Heating may cause a fire or explosion

H242 Heating may cause a fire

H250 Catches fire spontaneously if exposed to air

H251 Self-heating; may catch fire

H252 Self-heating in large quantities; may catch fire

H260 In contact with water releases flammable gases which may ignite spontaneously

H261 In contact with water releases flammable gas

H270 May cause or intensify fire; oxidizer

H271 May cause fire or explosion; strong oxidizer

H272 May intensify fire; oxidizer

H280 Contains gas under pressure; may explode if heated

H281 Contains refrigerated gas; may cause cryogenic burns or injury

H290 May be corrosive to metals

H300: Health hazards

H300 Fatal if swallowed.


58

H301 Toxic if swallowed

H302 Harmful if swallowed

H303 May be harmful if swallowed

H304 May be fatal if swallowed and enters airways

H305 May be harmful if swallowed and enters airways

H310 Fatal in contact with skin

H311 Toxic in contact with skin

H312 Harmful in contact with skin

H313 May be harmful in contact with skin

H314 Causes severe skin burns and eye damage

H315 Causes skin irritation

H316 Causes mild skin irritation

H317 May cause an allergic skin reaction

H318 Causes serious eye damage

H319 Causes serious eye irritation

H320 Causes eye irritation

H330 Fatal if inhaled

H331 Toxic if inhaled

H332 Harmful if inhaled

H333 May be harmful if inhaled

H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled

H335 May cause respiratory irritation

H336 May cause drowsiness or dizziness

H340 May cause genetic defects

H341 Suspected of causing genetic defects

H350 May cause cancer


59

H351 Suspected of causing cancer

H360 May damage fertility or the unborn child

H361 Suspected of damaging fertility or the unborn child

H361d Suspected of damaging the unborn child

H361e May damage the unborn child

H361f Suspected of damaging fertility

H361g may damage fertility

H362 May cause harm to breast-fed children

H370 Causes damage to organs

H371 May cause damage to organs

H372 Causes damage to organs through prolonged or repeated exposure

H373 May cause damage to organs through prolonged or repeated exposure

H300+H310 Fatal if swallowed or in contact with skin

H300+H330 Fatal if swallowed or if inhaled

H310+H330 Fatal in contact with skin or if inhaled

H300+H310+H330 Fatal if swallowed, in contact with skin or if inhaled

H301+H311 Toxic if swallowed or in contact with skin

H301+H331 Toxic if swallowed or if inhaled

H311+H331 Toxic in contact with skin or if inhaled

H301+H311+H331 Toxic if swallowed, in contact with skin or if inhaled

H302+H312 Harmful if swallowed or in contact with skin

H302+H332 Harmful if swallowed or if inhaled

H312+H332 Harmful in contact with skin or if inhaled

H302+H312+H332 Harmful if swallowed, in contact with skin or if inhaled

H303+H313 May be harmful if swallowed or in contact with skin

H303+H333 May be harmful if swallowed or if inhaled


60

H313+H333 May be harmful in contact with skin or if inhaled

H303+H313+H333 May be harmful if swallowed, in contact with skin or if inhaled

H315+H320 Causes skin and eye irritation

H400: Environmental hazards

H400 Very toxic to aquatic life

H401 Toxic to aquatic life

H402 Harmful to aquatic life

H410 Very toxic to aquatic life with long-lasting effects

H411 Toxic to aquatic life with long-lasting effects

H412 Harmful to aquatic life with long-lasting effects

H413 May cause long-lasting harmful effects to aquatic life

H420 Harms public health and the environment by destroying ozone in the upper atmosphere

H433 Harmful to terrestrial vertebrates

Country-specific hazard statements

Physical properties

EUH001 Explosive when dry

EUH006 Explosive with or without contact with air, deleted in the fourth adaptation to technical

progress of CLP.

EUH014 Reacts violently with water

EUH018 In use may form flammable/explosive vapour-air mixture

EUH019 May form explosive peroxides

EUH044 Risk of explosion if heated under confinement

Health properties

EUH029 Contact with water liberates toxic gas


61

EUH03 1Contact with acids liberates toxic gas

EUH032 Contact with acids liberates very toxic gas

EUH066 Repeated exposure may cause skin dryness or cracking

EUH070 Toxic by eye contact

EUH044 Risk of explosion if heated under confinement

Environmental properties

EUH059 Hazardous to the ozone layer, superseded by GHS Class 5.1 in the second adaptation

to technical progress of CLP.

Other EU hazard statements

EUH201 Contains lead. Should not be used on surfaces liable to be chewed or sucked by

children.

EUH201A Warning! Contains lead.

EUH202 Cyanoacrylate. Danger. Bonds skin and eyes in seconds. Keep out of the reach of

children.

EUH203 Contains chromium(VI). May produce an allergic reaction.

EUH204 Contains isocyanates. May produce an allergic reaction.

EUH205 Contains epoxy constituents. May produce an allergic reaction.

EUH206 Warning! Do not use together with other products. May release dangerous gases (chlorine).

EUH207 Warning! Contains cadmium. Dangerous fumes are formed during use. See information supplied by the manufacturer. Comply with the safety instructions.

EUH208 Contains <name of sensitising substance>. May produce an allergic reaction.

EUH209 Can become highly flammable in use.

EUH209A Can become flammable in use.

EUH210 Safety data sheet available on request.

EUH401 To avoid risks to human health and the environment, comply with the instructions

for use.


62

Precautionary statements

General precautionary statements

P101 If medical advice is needed, have product container or label at hand.

P102 Keep out of reach of children.

P103 Read label before use.

Prevention precautionary statements

P201 Obtain special instructions before use.

P202 Do not handle until all safety precautions have been read and understood.

P210 Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking.

P211 Do not spray on an open flame or other ignition source.

P220 Keep/Store away from clothing/…/combustible materials.

P221 Take any precaution to avoid mixing with combustibles.

P222 Do not allow contact with air.

P223 Do not allow contact with water.

P230 Keep wetted with …

P231 Handle under inert gas.

P232 Protect from moisture.

P233 Keep container tightly closed.

P234 Keep only in original container.

P235 Keep cool..

P240 Ground/bond container and receiving equipment.

P241 Use explosion-proof electrical/ventilating/lighting/…/equipment.

P242 Use only non-sparking tools.

P243 Take precautionary measures against static discharge.

P244 Keep valves and fittings free from oil and grease.

P250 Do not subject to grinding/shock/…/friction.

P251 Do not pierce or burn, even after use.

P260 Do not breathe dust/fumes/gas/mist/vapours/spray.

P261 Avoid breathing dust/fumes/gas/mist/vapours/spray.

P262 Do not get in eyes, on skin, or on clothing.

P263 Avoid contact during pregnancy/while nursing.

P264 Wash … thoroughly after handling. [… (to be specified)].

P270 Do not eat, drink or smoke when using this product.

P271 Use only outdoors or in a well-ventilated area.

P272 Contaminated work clothing should not be allowed out of the workplace.

P273 Avoid release to the environment.


63

P280 Wear protective gloves/protective clothing/eye protection/face protection.

P282 Wear cold insulating gloves/face shield/eye protection.

P283 Wear fire/flame resistant/retardant clothing.

P284 [In case of inadequate ventilation] wear respiratory protection.

P231+232 Handle under inert gas. Protect from moisture

P235+410 Keep cool. Protect from sunlight

Response precautionary statements

P301 IF SWALLOWED:

P302 IF ON SKIN:

P303 IF ON SKIN (or hair):

P304 IF INHALED:

P305 IF IN EYES:

P306 IF ON CLOTHING:

P308 If exposed or concerned:

P310 Immediately call a POISON CENTER/doctor/…

P311 Call a POISON CENTER/ doctor/…

P312 Call a POISON CENTER/ doctor/…/if you feel unwell.

P313 Get medical advice/attention.

P314 Get medical advice/attention if you feel unwell.

P315 Get immediate medical advice/attention.

P320 Specific treatment is urgent (see … on this label).

P321 Specific treatment (see … on this label).

P330 Rinse mouth.

P331 Do NOT induce vomiting.

P332 If skin irritation occurs:

P333 If skin irritation or a rash occurs:

P334 Immerse in cool water/wrap in wet bandages.

P335 Brush off loose particles from skin.

P336 Thaw frosted parts with lukewarm water. Do not rub affected areas.

P337 If eye irritation persists:

P338 Remove contact lenses if present and easy to do. Continue rinsing.

P340 Remove person to fresh air and keep comfortable for breathing.

P342 If experiencing respiratory symptoms:

P351 Rinse cautiously with water for several minutes.

P352 Wash with plenty of water/…

P353 Rinse skin with water/shower.

P360 Rinse immediately contaminated clothing and skin with plenty of water before removing clothes.


64

P361 Take off immediately all contaminated clothing.

P362 Take off contaminated clothing.

P363 Wash contaminated clothing before reuse.

P364 And wash it before reuse.

P370 In case of fire:

P371 In case of major fire and large quantities:

P372 Explosion risk in case of fire.

P373 DO NOT fight fire when fire reaches explosives.

P374 Fight fire with normal precautions from a reasonable distance.

P375 Fight fire remotely due to the risk of explosion.

P376 Stop leak if safe to do so.

P377 Leaking gas fire – do not extinguish unless leak can be stopped safely.

P378 Use … to extinguish.

P380 Evacuate area.

P381 Eliminate all ignition sources if safe to do so.

P391 Collect spillage.

P301+310 IF SWALLOWED Immediately call a POISON CENTER/doctor/…

P301+312 IF SWALLOWED Call a POISON CENTER/doctor/…/if you feel unwell.

P301+330+331 IF SWALLOWED Rinse mouth. Do NOT induce vomiting.

P302+334 IF ON SKIN Immerse in cool water/wrap in wet bandages.

P302+352 IF ON SKIN Wash with plenty of water/…

P303+361+353 IF ON SKIN (or hair) Take off immediately all contaminated clothing. Rinse skin with water/ shower.

P304+312 IF INHALEDCall a POISON CENTER or doctor/physician if you feel unwell.

P304+340 IF INHALED Remove person to fresh air and keep comfortable for breathing.

P305+351+338 IF IN EYES Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do – continue rinsing.

P306+360 IF ON CLOTHING Rinse immediately contaminated clothing and skin with plenty of water before removing clothes.

P308+311 If exposed or concerned Call a POISON CENTER/ doctor/…

P308+313 If exposed Call a POISON CENTER or doctor/physician.

P332+313 If skin irritation occurs Get medical advice/attention.

P333+313 If skin irritation or a rash occurs Get medical advice/attention.

P335+334 Brush off loose particles from skin. Immerse in cool water/wrap in wet bandages.

P337+313 If eye irritation persists get medical advice/attention.

P342+311 If experiencing respiratory symptoms Call a POISON CENTER/doctor/…

P361+364 Take off immediately all contaminated clothing and wash it before reuse.

P362+364 Take off contaminated clothing and wash it before reuse.

P370+376 In case of fire Stop leak if safe to do so.

P370+378 In case of fire Use … to extinguish.


65

P370+380 In case of fire Evacuate area.

P370+380+375 In case of fire Evacuate area. Fight fire remotely due to the risk of explosion.

P371+380+375 In case of major fire and large quantities Evacuate area. Fight fire remotely due to the risk of explosion.

Storage precautionary statements

P401 Store …

P402 Store in a dry place.

P403 Store in a well ventilated place.

P404 Store in a closed container.

P405 Store locked up.

P406 Store in a corrosive resistant/… container with a resistant inner liner.

P407 Maintain air gap between stacks/pallets.

P410 Protect from sunlight.

P411 Store at temperatures not exceeding … °C/… °F.

P412 Do not expose to temperatures exceeding 50 °C/122 °F.

P413 Store bulk masses greater than … kg/… lbs at temperatures not exceeding … °C/… °F.

P420 Store away from other materials.

P422 Store contents under …

P402+404 Store in a dry place. Store in a closed container.

P403+233 Store in a well ventilated place. Keep container tightly closed.

P403+235 Store in a well ventilated place. Keep cool.

P410+403 Protect from sunlight. Store in a well-ventilated place.

P410+412 Protect from sunlight. Do not expose to temperatures exceeding 50 °C/122 °F.

P411+235 Store at temperatures not exceeding … °C/… °F. Keep cool.

Disposal precautionary statements

P501 Dispose of contents/container to … [… in accordance with local/regional/national/international regulation (to be specified)].

P502 Refer to manufacturer/supplier for information on recovery/recycling.

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