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

### Kol n

Germany

Date: 02 Apr il 2012

### LNX33444 A Spect r al Service A G Study Report

Emii-Hoffmann-StraBe 33 D-50996 Koln (Germany) page 2 o f 16 TABLE OF CONTENTS

1 OBJECT I VES

............................................................................................................................... ............. 4

2 TEST IT EM

.............................................................................................................................. ................. 4

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

3.1 Reference and Calibration Ite ms .................................................................................................... 4

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

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

4 METHODS

.............................................................................................................................. ................. 5

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

4.2 Ma ss S pectros copy......................................................................................................................... 5

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

6 SUMMARY

.............................................................................................................................. ............... 16

7 PERSONNEL

.............................................................................................................................. ........... 16

8 CONFIRMATION OF T HE STUDY REPORT ......................................................................................... 16

### LNX33444 A Spect r al Service A G Study Report

Emii-Hoffmann-Strat1e 33 D-50996 Koln (Germany) page 3 of 16 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

±ES I/M S of the reaction product of test item LNX33444 with serine ............................................. 8

+ES I/MS of the reaction product of the test item LNX33444 with ser i ne ....................................... 8

13

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

13

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

13

C

- 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

±E SI /MS of the reaction product of test item LNX33444 with l ysine ............................................ 12

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

HMBC spectrum of the test item LNX33444 after reaction with lys i ne ........................................ 13

13

C

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

13

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

13

C

- NMR spectra overlay of test item LNX33444 during th e reaction with lysine, Carbonyl

group signal .........

............... .............. ............... ................. ............... .............. ............... ................ 15

13

C

- 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 ass ignment -E SI mass spectrum and ±E S I mass spectra ..................................................... 7

Peak assignment -E SI mass spectrum and ±E S I mass spec tra ................................................... 11

### LNX33444 A Spect r al Service A G Study Report

Emii-Hoffmann-Strar1e 33 D-50996 Koln (Germany) page 4 of 16 1 OBJECTIVES

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

conditions

.

2 TEST I TEM

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 it ems

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 A G Study Report LNX33444_A

Emii-Hoffmann-Stra e 33 D-50996Koln(Germany) page 5 of 16 3.3 Instruments

NMR spectrometer Avance Ill 600 (Bruker, Karlsruhe, D), magn etic 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)

Comp uter 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 M ET HOD S

4.1 NMR Spectroscopy

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

dissolved i n D 2 

### 0 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 13 C-N M R spectra have been recorded to

characterise the reaction products.

The actually used NMR parameters appear on the spectrum plot.

4.2 Mass Spe ctroscopy

±ESI mass spectra were recorded to characterise the derivatised test i tem. 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

### Emii-Hoffmann-Stra e 33 D-50996Koln(Germany) page 6 of 16 Spectral Service A G Study Report LNX33444 A

5 RESULTS AND DISCUSSION

The react i on between serine and X-Tan is a competitive reaction to the i ntra molecular po l ymeri

zation

. The end product is a molecule of peptide structure (cf. Fig

. 3)

.

NaOH

OHO /N H

, 

# a=\_

)-N H

NH Fonnula Weight 

## = 247.29144

OH

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

HO O

2 H2N OH

0

NaOH t OH

NH

NH -{

OHO

1 2

5 :0 1

62

OH : ula We<Jht 

### = 378.3 78 24

Fig. 3 Possible reaction product of X-Tan with two serine molec ul es (M2)

### Spectral Service A G S tudy Report LNX33444_A

Emii-Hoffmann-StraBe 33 D-50996 Koln(Germany) page 7 of 16 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 S ignal assignment +ESI m/z Signal assignment

- ESI

248 [M1+Ht 188 [M2-2Hr

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

399 [M2+Na -2 H)"

### Spectral Service A G Study Report 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

s i d=10,00 Full ms (

50,00 -11 00

, 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 1 009,1

LNX_XTanSerines i#11-3 0

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 50 0

m/z 600 700 800 830,8

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



### Emii-Hoffmann-Stra e 33 D-50996 Koln (Germany) page 8 of 16**NN,.._omcnco(O&n&n'¢ c:icioicricO,...:,...:.ou;..nu->** 

**-.:t"':J"MNNNNNNNN** 

### \)

50 40 30 20 1 0 m ' I " 'I " I "' 1 "" 

# 1 11

,_,..... 

# 1

. .. 1 ' 

# 1 1 ! § 1 1

...,... , 190 180 110 160 150 140 130 12 0 1 10 100 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 13 C-NMR spectrum of test i tem LNX33444 w i th ser i ne at pH 5

:T

AN+ Serin pH 1•



### ....,..... . ... . - ·· ....-o-r .........., ,.,.,

1 90 180 170 160 1 50 140 130 120 110 1 00 90 80 70 60

F ig. 7 13 C-NMR spectrum of test item LNX33444 w i th serine a t pH 1 0

, start of the reac t ion



### Emii-Hoffmann-StraBe 33 D-50996 Koln (Germany) page 9 of 16

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

•.

' '



# 1 1 

# ( 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 1 1 0 100 90 80 70 60 50 40 3 0 20 10

Fig. 8 13 C-NMR spectrum of test ite m LNX33444 with se rine 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 +E S I m/z Signal assignment -ESI

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

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

527 [M+3Na-2H]"'"

### Spectral Service A G Study Report LNX33444 A

Emii-Hoffmann-StraBe 33 D-50996 Koln (Germany) page 12 o f 16 Dc 1

Dat

eniL NX

_ XTanLystnes 1

V 1.2) AOA. Sprltzenpompe

6.2 2012 1 0:2 4 :29 L NX - XTan X-T a n + Lysll

65,4 LNX_XTanLysinesi #11

- 35

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

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

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 :0,3 1-0,8 0 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:,oat e n\L NX

_ XTanlysinesop

V 1.2) A OA: S p rotzenporrope 06.02.2012 1 0:25: 4 7 L NX- 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

1 0

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 Se r vice A G Study Report LNX33444 A

Emii-Hoffmann-StraBe 33 D-50996 Koln (Germany) page 13 o f 16 

# . , . . .

'

### Spectral Service A G Study Report LNX33444 A

Emii-Hoffmann-Stral1e 33 D-50996 Koln (Germany) page 14 of 16 XT AN + Lysin Tage + NaOD



### Ly s in e

CO amino acid Molecule head

- linked

Molecule tail

- linked 

### CO urea

1 88 1 86 1 84 18 2 180 178 1 76 174 172 170 168 166 164 16 2 1 6 0 ppm

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

detail, carbonyl region

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

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 va lue and the reaction time. This signal

dynamic enables on the one hand the monitoring of the actual pH va lue dur ing the reaction and on

the other hand the determination of the reaction kinetics

.

### Spectral Service A G Study Report LNX33444

_ A

3l 101 E: TO P SPIJI D1.e:hl

!kelc : 16.00

33 10 0 E: \ TO P P I N D1 ehl

cole : J6.Ct0

E : 1t pn: 1.1 •

e a1 35

. 291

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

176

. 0 175

. 5 175

. 0 17 4 .5 (pp m )

Fig. 15 1 3 C-NMR spectra overlay of test item LNX33444 during the reaction with l ys in e

, Carbonyl group signal

TO P$UJI I> U

I •l•

1 16 C

176

. 0 17 5

. 5 17 5 .0 17 4

. 5 ( ppm )

Fig. 16 1 3 C-NMR spectra of test i tem LNX33444 during the reaction with lys in e

, Carbonyl group signal



### Emii-Hoffmann-Stra e 33 D-50996 Koln (Germany) page 1 5 of 16

### Spect r al Se r vice A G Study Report LNX33444 A

6 SUMMARY

Amino acids and amines react with the test i tem X-tan at high pH values to urea der i vatives. The

reaction w i t h the a-amino group of am i no acids surprisingly reacts f aster than the terminal amino

group of a l kyl amide

. This cou l d be demonstrated especially i n case of lysine

, which consists of both

types of am in o groups.

The results of the present study can most probably be extrapolated to a ll amino acids

, and especially

to pept i des 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

Biologica l technical assistant

Food chemist

Laboratory assistant

Laboratory assistant

All are staff members of the test fac i lity

.



### 8 CONF I RMAT I ON OF THE STUDY REPORT

Date : 02 April 2012 Company stamp:

Study director: 

# Spectral 

# Service P G

Err.i i -Hoffmann-Str. 33

· D-50 995 

### r <oln

0 22 36/9 69 47-0

· Fa x 0 22 36/9 69 4 i·1 1



### www .spectralservice .de

Page 1 o f t he original report i s pr i nted on Spectral Service business paper ; the report i s authorised

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

and is not a ll owed to be connected w i th the company's name or a signature of a staff member

.

M i suse will be prosecuted.



### Emii-Hoffmann-Strar1e 33 D-50996 Koln (Germany) page 1 6 of 1 6