Emii-Hoffmann-Stra e 33 D-50996 Koln (Germany) page 1of 16

Spectral Service

STUDY REPORT LNX33444 A

- X-tan pure (RJE76081)-

- Reaction with Amino Acids -

Sponsor: Lanxess Deutschland GmbH

lEA Regulatory Affairs & Product safety

Chem. Park Leverkusen Geb. 19

D-51369 Leverkusen

Germany

Monitor: Dr. Qin Zhu-Ohlbach

Order no.: 310412397

Test Facility: Spectral Service AG

Emii-Hoffmann-StraBe 33

D-50996 Koln

Germany

Date: 02 Apr il 2012
Spectral Service AG Study Report LNX33444 A

Emii-Hoffmann-StraBe 33 D-50996 Koln (Germany) page 2 of 16

TABLE OF CONTENTS

1 OBJECT IVES............................................................................................................................................ 4

2 TEST ITEM ............................................................................................................................................... 4

3 MATERIALS ............................................................................................................................................. 4

3.1 Reference and Calibration Items .................................................................................................... 4

3.2 Chem icals and Materials ................................................................................................................ 4

3.3 Instruments ..................................................................................................................................... 5

4 METHODS ............................................................................................................................................... 5

4.1 NMR Spectroscopy ......................................................................................................................... 5

4.2 Mass Spectroscopy......................................................................................................................... 5

5 RESULTS AND DISCUSSION ................................................................................................................. 6

6 SUMMARY ............................................................................................................................................. 16

7 PERSONNEL ......................................................................................................................................... 16

8 CONFIRMATION OF THE STUDY REPORT ......................................................................................... 16
Spectral Service AG Study Report LNX33444 A

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FIGURES

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

Fig. 12

Fig. 13

Fig. 14

Fig. 15

Fig. 16

TABLES

Tab. 1

Tab.2

Tab. 3

Tab. 4

Tab.5

Chemical structure of the test item ................................................................................................ 4

Possible reaction product of X-Tan with one serine molecule (M1) ............................................... 6

Possible reaction product of X-Tan with two serine molecules (M2) .............................................. 6

±ESI/MS of the reaction product of test item LNX33444 with serine ............................................. 8

+ESI/MS of the reaction product of the test item LNX33444 with serine ....................................... 8

13C-NMR spectrum of test item LNX33444 with serine at pH 5 ..................................................... 9

13C-NMR spectrum of test item LNX33444 with serine at pH 10, start of the reaction ................... 9

13C-NMR spectrum of test item LNX33444 with serine at pH 10 after the reaction was finished10

Possible reaction product of X-Tan with lysine (formal) ............................................................... 11

±ESI/MS of the reaction product of test item LNX33444 with lysine ............................................ 12

+ESI/MS of the reaction product of test item LNX33444 with lysine ........................................... 12

HMBC spectrum of the test item LNX33444 after reaction with lysine ........................................ 13

13C-NMR spectrum of the test item LNX33444 after reaction with lysine .................................... 13

13C-NMR spectrum of the test item LNX33444 after reaction with lysine, detail, carbonyl region 14

13C-NMR spectra overlay of test item LNX33444 during the reaction with lysine, Carbonyl

group signal .................................................................................................................................. 15

13C-NMR spectra of test item LNX33444 during the reaction with lysine, Carbonyl group signal 15

Test item data ................................................................................................................................ 4

Chemicals actually used as reference and calibration items ......................................................... 4

Chemicals and materials used in the study ................................................................................... 4

Peak assignment -ESI mass spectrum and ±ESI mass spectra ..................................................... 7

Peak assignment -ESI mass spectrum and ±ESI mass spectra ................................................... 11
Spectral Service AG Study Report LNX33444 A

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1 OBJECTIVES

The purpose of analysis is to develop the reaction of X-Tan with amino acids under hydrolysis

conditions .

2 TEST ITEM

Tab. 1 Test item data

Sample name Lot no. Spectral Service code Arriva l

X-tan pure (RJE76081) - LNX33444-1 17.02.2012 

Fig. 1 Chemical structure of the test item

3 MATERIALS

3.1 Reference and Calibration Items

Tab.2 Chemicals actually used as reference and calibration items

No Substance name Distributor Order no.

15 3-(Trimethylsilyl}-propionic acid-d4 Na-salt 

(TMSP; for NMR calibration}

E. Merck

Darmstadt (D) 8652

3.2 Chemicals and Materials

Tab.3 Chemicals and materials used in the study

Substance name Distributor Order no.

Deuteriumoxide, Degree of deuteration 99.9% Euriso-top, Saarbriicken (D) D214H

Dimethylsulfoxide-ds (DMSO-ds} ,

Degree of deuteration 99.9%

Euriso-top, Saarbruc ken (D) D031H

DL-Serine Fluka Chemie AG (D) 84980

L-Lysine Fluka Chemie AG (D) ---
Spectral Service AG Study Report LNX33444_A

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3.3 Instruments

NMR spectrometer Avance Ill 600 (Bruker, Karlsruhe, D), magnetic flux density 14.1 Tesla

QNP cryo probe head; automated sample changer Bruker B-ACS 120

Computer Intel Core2 Duo 2.4 GHz under MS Windows XP and Bruker TopSpin 2.1 for acquisition

Bruker TopSpin 2.1 for processing

Standard operation procedure SAA-GMR028-02

Mass spectrometer Finnigan AQA (ThermoQuest, Manchester, GB)

±ESI modus, introduction via syringe pump

Syringe pump Harvard Apparatus 11 Plus (March, D)

Computer Pill 500MHz under Windows NT 4.0 and Software Xcalibur 1.2

Standard operation procedure SAA-GMR01 0-03

Electronic semi-micro balance Sartorius R 180 D-*D1 (Gottingen, D)

Balance printer Sartorius YDP03-0CE (Gottingen, D)

Standard operation procedure SAA-GMR005-04

4 METHODS

4.1 NMR Spectroscopy

The test item LNX33444 (X-Tan) was combined with amino acids serine and lysine, respectively, and

dissolved in D20 in a ratio of 1:2. Sodium hydroxide solution was added in small amounts step by

step in order to initiate the reaction. 1

H-NMR spectra and 13C-NMR spectra have been recorded to

characterise the reaction products.

The actually used NMR parameters appear on the spectrum plot.

4.2 Mass Spectroscopy

±ESI mass spectra were recorded to characterise the derivatised test item. The NMR solution was

directly introduced via syringe pump.

Scan parameter:

Capillary voltage:

Source voltage:

Probe temperature:

50-1100 amu in 2.9 sec.

4 kV

+10/-10V

300°C
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Spectral Service AG Study Report LNX33444 A

5 RESULTS AND DISCUSSION

The reaction between serine and X-Tan is a competitive reaction to the intra molecular polymerization.The end product is a molecule of peptide structure (cf. Fig. 3).

NaOH

OHO /NH,

a=\_ )-NH

NH Fonnula Weight = 247.29144

OH

Fig. 2 Possible reaction product of X-Tan with one serine molecule (M1)

HO O

2 H2N OH

0

NaOH tOH

NH

NH-{

OHO

1 2

5 :01

62

OH

: ula We<Jht = 378.37824

Fig. 3 Possible reaction product of X-Tanwith two serine molecules (M2)
Spectral Service AG Study Report LNX33444_A

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The mass spectra show a quasi-molecular ion [M+H)" at m/z 377 (cf. Fig. 4 and Fig. 5). The adduct

ions and cluster (cf. Tab. 4) are in accordance with the proposed structures of the react products (cf.

Fig. 2 and Fig. 3).

Tab.4 Peak assignment -ESI mass spectrum and ±ESI mass spectra

m/z Signal assignment +ESI m/z Signal assignment -ESI

248 [M1+Ht 188 [M2-2Hr

270 [M1+Nat 377 [M2-H)"

    399 [M2+Na-2H)"
Spectral Service AG StudyReport LNX33444 A

60

0:\0aten\LNX XTanSeronesi

v .1.2) A OA; Sprrtzenpumpe

06.02.2012 10:28:25 LNX- XTan X·Tan + Seron 

c:

ltl

100

80

270,1 LNX_XTanSerinesi#1 0-34

RT:0,29-0,82 A V: 12 NL:

1,44E5 F:{0;0} + c ESI

sid=10,00 Full ms (

50,00-1100,00]

"

c

0

::::J

.0

<(

(I)

:

>co 40

ltl

Qi

128,2 271•

2 412 ,3

0:: 20

0

3,8

3,0

2,5

2,0

188,5

378,2

1009,1

LNX_XTanSerines i#11-30

RT: 0,31-0,75 AV : 10 NL:

5,44E3 F :{0;1}- c ESI

sid=10,00 Full ms [

50,00-1100,00]

1,5 246,2 399,2

200 400 600

m/z

800 1000

Fig. 4 ±ESIIMS of the reaction product of test item LNX33444 with serine

0:\Daten\LNX_XTanSeronesip 

V.1.2) AQA; Sprrtzenpu"1>e

06.02.2012 10:29: 2 LNX- XTan X·Tan + Seron

LNX_XTanSerinesip #5-23 RT: 0,19-0,79 AV: 19 NL: 2,47E5

T: {0;0} + c ESI sid=10,00 Full ms [ 50 ,00-1630 ,00]

100 270,2

90

80

70

(I) 0

c:

ltl 60

"

c

0

:::J:

.0

<( 50 (I)

> 121,2

Qi 40

0:: 128,1 271,3

30 412,4

20 248 ,3 390,3 445,4

177,1

10

0

100 200 300 400 500

m/z

600 700 800

830,8

Fig. 5 +ESI/MS of the reaction product of the test item LNX33444 with serine

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NN,.._omcnco(O&n&n'¢

c:icioicricO,...:,...:.ou;..nu->

-.:t"':J"MNNNNNNNN

\)

    

50 40 30 20 10 m

'I" 'I" I"' 1 ""

1 11 ,_,.....1 . ..1'

1 1 !§1 1 ...,..., 190 180 110 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10

Spectral Service AG Study Report LNX33444_A 

X-Tan + Serin 1/2 c"..'

.

i

I

0,.>.:L"

t)'

"

\

'"I'

r-.........,.-

Fig. 6 13C-NMR spectrum of test item LNX33444 with serine at pH 5

 :TAN+ Serin pH 1•

....,.......... - ·· ....-o-r .........., ,.,., 190 180 170 160 150 140 130 120 110 100 90 80 70 60

Fig. 7 13C-NMR spectrum of test item LNX33444 with serine at pH 10, start of the reaction

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Emii-Hoffmann-Strar.,e 33 D-50996 Koln (Germany) page 10 of 16

,.

..•.

''

 11 ( 11

Spectral Service AG Study Report LNX33444 A 

0

co

XTAN + Serin Tage + NaOD

co 0,....,.... a) a)U')t.ri (") NNN

I \ V

.,_...

;

- , ... 11; ;· .. ·. ··· Y

.

. 

,..... ,

'·,.'

, ....

....,......-.,.·· · ···..,.---r··· ,.........,...........,. ··· 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10

Fig. 8 13C-NMR spectrum of test item LNX33444 with serine at pH 10 after the reaction was

finished
Spectral Service Study Report LNX33444_A

Emii-Hoffmann-Stra e 33 D-50996Koln(Germany) page 11 of 16

OH

A more complex reaction occurs between X-Tan and the amino acid lysine, as it has two amino

groups. Both amino groups react to a hypothetic molecule (cf. Fig. 9). The amino group showed a

higher affinity to form a peptide than pure urea.

0 PH

:::::s ::::: NH-{ O

0

0

0 O }-NH

2 :::::s,.... I "'0

HO

NaOH

OH 0

0 }-NH

NH

Formula Weight = 460.5682

Fig. 9 Possible reaction product of X-Tan with lysine (formal)

The mass spectra show a quasi-molecular ion [M-Hr at m/z 459 (cf. Fig. 10 and Fig. 11). The adduct

ions are in accordance with the proposed structure of the reaction product (cf. Fig. 9).

Tab.5 Peak assignment -ESI mass spectrum and ±ESI mass spectra

m/z Signal assignment +ESI m/z Signal assignment -ESI

483 [M+Na]"'" 459 [M-Hr

505 [M+2Na-H]"'"    

527 [M+3Na-2H]"'"    
Spectral Service AG Study Report LNX33444 A

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Dc 1DateniLNX _XTanLystnes 1

V 1.2) AOA. Sprltzenpompe

6.2 

2012 10:24:29 LNX- XTan X-Tan + Lysll

65,4 LNX_XTanLysinesi#11 -35

RT: 0,30-0,87 AV: 13 NL:

2,08E5 F: {0;0} + c ESI

sid=10,00 Full ms [

50,00-1100,00]

169,3

460,4

653•5 819,2 923,5 1003,1

LNX_XTanLysinesi#12-32

RT:0,31-0,80 AV: 11 NL:

4,28E3 F: {0;1} - c ESI

sid=10,00 Full ms [

50,00-1100,00]

245,1

033,9

m/z

Fig. 10 ±ESI/MS of the reaction product of test item LNX33444 with lysine

0:,oaten\LNX_XTanlysinesop

V 1.2) AOA: Sprotzenporrope

06.02.2012 10:25:47 LNX- XTan X-Tan+ Lysin

LNX_XTanLysinesip #5-26 RT: 0,19-0,89 AV : 22 NL: 3 ,01E5

T: {0;0} + c ESI sid=10,00 Full ms [ 50,00-1630,00]

100 65,3

90

80

70

Q)

0

c(1) 60

"

c

0

:l

.0

<{ 50 Q)

>

Qi 40

a::

30

121,2

89,3

169,1

505 ,3

20 289,3 483,4 527,3

191'1

528,3

10

0

871,9

100 200 300 400 500

m/z

600 700 800

Fig. 11 +ESI/MS of the reaction product of test item LNX33444 with lysine
Spectral Service AG Study Report LNX33444 A

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.

,...

 

' 
Spectral Service AG Study Report LNX33444 A

Emii-Hoffmann-Stral1e 33 D-50996 Koln (Germany) page 14 of 16

 

XT AN + Lysin Tage + NaOD

Lysine

CO amino acid Molecule head-linked

Molecule tail-linked

CO urea

188 186 184 182 180 178 176 174 172 170 168 166 164 162 160 ppm

Fig. 14 13C-NMR spectrum of the test item LNX33444 after reaction with lysine,

detail, carbonyl region

The reaction of X-Tan with lysine could be monitored in 13C-NMR spectra due the chemical shifting

of the amino group signal of lysine from 175.8 ppm to 174.7 ppm (cf. Fig. 15 and Fig. 16).

The chemical shift of this signal correlated with the pH value and the reaction time. This signal

dynamic enables on the one hand the monitoring of the actual pH value during the reaction and on

the other hand the determination of the reaction kinetics.
Spectral Service AG Study Report LNX33444_A

   

3l 101 E: TOPSPIJI D1.e:hl

!kelc : 16.00

33 100 E: \TOP PIN D1ehl

cole : J6.Ct0

E : 1t pn: 1.1 •

ea1 35.291

11 1 1::\TOP:SPDI D:tehl

176.0 175.5 175.0 174.5 (ppm)

Fig. 15 13C-NMR spectra overlay of test item LNX33444 during the reaction with lysine,

Carbonyl group signal

TO P$UJI I>U

I •l• 1 16 C

176.0 175.5 175.0 174.5 (ppm)

Fig. 16 13C-NMR spectra of test item LNX33444 during the reaction with lysine,

Carbonyl group signal

Emii-Hoffmann-Stra e 33 D-50996 Koln (Germany) page 15 of 16
Spectral Service AG Study Report LNX33444 A

6 SUMMARY

Amino acids and amines react with the test item X-tan at high pH values to urea derivatives. The

reaction with the a-amino group of amino acids surprisingly reacts faster than the terminal amino

group of alkyl amide . This could be demonstrated especially in case of lysine, which consists of both

types of amino groups.

The results of the present study can most probably be extrapolated to all amino acids, and especially

to peptides on the surface of leather products.

7 PERSONNEL

Study director:

Co-worker:

Quality assurance:

Dr. Bernd Diehl,

Andreas Beyer,

Karen Gedig,

Birgit Hans, 

Sabine Holthausen,

Chemist

Biologicaltechnical assistant

Food chemist

Laboratory assistant

Laboratory assistant

All are staff members of the test facility.

8 CONFIRMATION OF THE STUDY REPORT

Date: 02 April 2012 Company stamp:

Study director: Spectral Service P G

Err.ii-Hoffmann-Str. 33 ·D-50995 r<oln

0 22 36/9 69 47-0 ·Fa x 0 22 36/9 69 4i·11

www .spectralservice .de

Page 1 of the original report is printed on Spectral Service business paper; the report is authorised

by original signature and company 's stamp. Use or publication in parts is not authorised on principle

and is not allowed to be connected with the company's name or a signature of a staff member.

Misuse will be prosecuted.

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