Us 20110311505

US 20110311505A1
(19) United States

(12) Patent Application Publication (10) Pub. N0.2 US 2011/0311505 A1
Ersoz et a].
(54)
(43) Pub. Date:
Dec. 22, 2011
PHOTOSENSITIVE AMINOACID-MONOMER
C08J 3/28
(2006.01)
LINKAGE AND BIOCONJUGATION

APPLICATIONS IN LIFE SCIENCES AND
BIOTECHNOLOGY
C07K 1/107
C07D 209/20
C07C 235/78
(2006.01)
(2006.01)
(2006.01)
Arzu Ersoz; Eskisehir (TR); Deniz

Hur; Eskisehir (TR); Filiz Yilmaz;
Eskisehir (TR); Adil Denizli;
C07C 323/59
A61P 35/00
C07F 17/00
B05D 5/06
(2006.01)
(200601)
(200601)
(200601)
Eskisehir (TR); Ayca Atilir Ozcan,
B82Y40/00
(201 1.01)
Eskisehir (TR); Sibel Emir

Diltemiz; Eskisehir (TR); Suzan
3821 5/00
B82Y 15/00
(201101)
(2011.01)
(75) Inventors:
(73) Assigneez
Yfzar Eslilseh. (TR); Qzlem
(52) us. Cl. ..... .. 424/943; 427/157; 427/214; 435/7.23;
B1e">E.Sk1SeP1r<TR>; .sbel
Byktyak.1ESk1.Seh1F (TR);
435/18; 435/19; 435/25; 435/28; 435/29;

435/188; 436/501; 436/86; 522/87; 530/364;
Rustem Keclh E.skls?h (TR);

Gun" s.ak":ESk1S?h1r.(TR);
Tugba Fmdk ESk1Seh1r(TR)
Ridvan say Eskisehir (TR)
530/380; 530/391 .1; 530/391.3; 530/400;

530/408; 530/409; 548/496; 562/445; 562/556;
204/157.75; 204/157.71; 977/746; 977/773;
(21) Appl. No.:
977/847; 977/896; 977/915; 977/920; 977/810
13/203,833
(57)
This invention is related to preparation of photosensitive

ruthenium based aminoacid monomers and oligomers; ami
(22)
PCT Filed:
Dec- 11: 2009
(86)
PCT N05
PCT/IB2009/055707
ABSTRACT
noacid monomer-protein cross-linking using photo sensitat

ion and conjugation on micro and nano-structures by ruthe
nium-chelate based monomers. Its vast range biotechnolgy
371 (6)0)
(2) (4) Date:
Aug 29 2011
_
Pubhcatlon Classl?catlon
applications of multifunctional; biocompatible; stabilE and

speci?c micro and nanobio-conjugates; Which Will stand
alone or simultaneously enable (i) both puri?cation and deter
(200601)
mination; (ii) both targeting and imaging and theranostics and

(iii) catalysis and determination. The construction and
method of preparation is applicable to silica materials; super
B05D 7/00
(200601)
paramagnetic particles; QDs; CNTs; Ag/Au nanoparticles
G01N 33/566
(200601)
and Au surfaces and polymeric materials. The photosensitive
C12Q 1/34
C12Q 1/44
(200601)
(200601)
aminoacid monomer linkers can react via chemically and

biocompatible to a lot of different micro and nano-surface and
C12Q 1/26
(200601)
then to the protein When they act as a single-step cross-linking
C12Q 1/28
(200601)
reaction using irradiation. The photosensitive conjugation
C12Q 1/02
(200601)
based on click biochemistry can be carried out at mild con
C12N 9/96
G01N 21/75
(200601)
(200601)
ditions; independent of pH and temperature; Without affect

ing conformation and function of protein.
(51) Int, Cl,

A61K 38/44
Patent Application Publication
Dec. 22, 2011 Sheet 1 0f 21
US 2011/0311505 A1
Figure 1
Figure 2
Dec. 22, 2011 Sheet 2 0f 21
US 2011/0311505 A1
Figure 3
OH
Figure 4
OH
OH
Figure 5
Dec. 22, 2011 Sheet 3 0f 21
US 2011/0311505 A1
Figure 6
Figure 7
Dec. 22, 2011 Sheet 4 0f 21
US 2011/0311505 A1
Figure 8
Figure 9
Dec. 22, 2011 Sheet 5 0f 21
US 2011/0311505 A1
Vs-Intensity
ZUEM
1.00 212.03
%Intens
QUE-1H4
10
MBSSWZJ
4039 4
8029.8
1117.6
12020.2
Mass (111.12]
16010.6
Dec. 22, 2011 Sheet 7 0f 21
US 2011/0311505 A1
1000 i
9@2.85
O35 671 6.
2 803 95
0.
52
1,
41
m
w 08,
42861
m
w
40O600
WW
6
5
0
m
i
7\O O
Ve e
F O] g u vl e l 5
W"
i
0
m
m
W
iV
11.O..hL
M
(e5
nw I
w
2.5:8
400
Wavelength (nm)
Figure 16
Dec. 22, 2011 Sheet 8 0f 21
US 2011/0311505 A1
Figure 17
\.
Figure 18
Dec. 22, 2011 Sheet 9 0f 21
US 2011/0311505 A1
50 0
4O 0
3i
51. 555 -
255 -
:2
E
190 _
.52, 55.455
O -'_':__4
'
30 0
Inn-bu"
4O 0
22%|---------- nwd
5O 0
60 0
7 U0
Wavelength (nm)
Figure 19
BUD
3 455
53
R
8.
.5
E 255
5
5'
\1
*\
U '5'
j f_,--?r35555,
I'm-M-15.554
H
fl =
,1
f;
555
455
rl|551 .52, 125.555

.
)1 "' (I
555
555
WWI
ml]
Wavelength (nm)
Figure 20
600
3 455
52
5
g 200
2'
i4~v4555215235
g?)
Wavelength (nm)
Figure 21
Dec. 22, 2011 Sheet 10 0f 21
US 2011/0311505 Al
600*
365:2V5
400
ETwNWmaHs.?M
A,89%m wmw,
3 3 S 0. 0 2 5 0 7. 2 3
200*
58102151840
300
400
500
600
700
Wavelength (nm)
Figure 22
mum w
Masai:
Figure 23
Dec. 22, 2011 Sheet 11 0f 21
US 2011/0311505 A1
Figure 24
50
+ Nanoparticles
& Free llpase
Figure 25
Dec. 22, 2011 Sheet 12 0f 21
US 2011/0311505 A1
+Free lipase
Nanoparticles
Temperature
Figure 26
350 i
300 i
200 i
35:2
150 i
100 *
50
10
12
Ma nnose Concentration (ppm)
Figure 27
Dec. 22, 2011 Sheet 13 0f 21
US 2011/0311505 A1
2%
2 00
1
w
1
w
lgM Concentration (ppm)
Figure 28
200
b
.1
25.:985
amahm
m apaCVVh.1t .n@
.mn
ninP
1mmdb
1 e6 s
a
.m
.w
5PuPI5afa0infin
5O
O0
n.w
w
MW
_
O
aw
6blom0tv
l5
6 0
l0
7 0
h Wm
Figure 29
Dec. 22, 2011 Sheet 14 0f 21
US 2011/0311505 A1
1 OO O
antitubulin antibody nanoparticles - <

'7
800 _
ccllfraction interaction at 0C
5:
600 _
P5,
5
antitubulin antibody nanopaiticles in
phosphate buffer
400 -
E
200 -
550
600
650
700
Wavelength (nm)
Figure 30
600
antiSOD-SPN in
/\
phosphate buffer
- 400
/ \/
"Z?
//'\\\
antiSOD-SPN + SOD
E 200
0 i
450
fraction
/i
\
i
\\
500
550
Wavelength (nm)
Figure 3 l
Dec. 22, 2011 Sheet 15 0f 21
US 2011/0311505 A1
600 i
500 i
400 i
Emis on
300 i
200 i
100*
5E-08
1E-07
1.5E-O7
2E-O7
2.5E-07
3E-07
C (M)
Figure 32
3.00E+10 *
2.70E+10 *
2.40E+10 *
2.10E+10 *
1.80E+10 *
1.50E+10
1.20E+10 *
9.00E+09 *
6.00E+09 *
3.00E+09 *
0.00E+00
200
300
400
500
600
700
Figure 33
Dec. 22, 2011 Sheet 16 0f 21
US 2011/0311505 A1
Figure 34
Figure 35
II
Dec. 22, 2011 Sheet 17 0f 21
US 2011/0311505 A1
CAu/Ag NC
5, 600
9 400
E
200
600
620
640
660
680
700
Wavelength (nm)
Figure 36
0.0000000.0000010.0000010.0000020.0000020.000003
C {M}
Figure 37
Dec. 22, 2011 Sheet 18 0f 21
US 2011/0311505 Al
w .
5qua? 1%
.v.
Figure 38
Figure 39
Dec. 22, 2011 Sheet 19 0f 21
US 2011/0311505 A1
Figure 40
Figure 41

Us 20110311505

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(19) United States

(43) Pub. Date:

Dec. 22, 2011

LINKAGE AND BIOCONJUGATION

Arzu Ersoz; Eskisehir (TR); Deniz

Eskisehir (TR); Ayca Atilir Ozcan,

Eskisehir (TR); Sibel Emir

Yfzar Eslilseh. (TR); Qzlem

(52) us. Cl. ..... .. 424/943; 427/157; 427/214; 435/7.23;

435/18; 435/19; 435/25; 435/28; 435/29;

Rustem Keclh E.skls?h (TR);

530/380; 530/391 .1; 530/391.3; 530/400;

(21) Appl. No.:

977/847; 977/896; 977/915; 977/920; 977/810

This invention is related to preparation of photosensitive

Dec- 11: 2009

noacid monomer-protein cross-linking using photo sensitat

applications of multifunctional; biocompatible; stabilE and

mination; (ii) both targeting and imaging and theranostics and

paramagnetic particles; QDs; CNTs; Ag/Au nanoparticles

and Au surfaces and polymeric materials. The photosensitive

aminoacid monomer linkers can react via chemically and

then to the protein When they act as a single-step cross-linking

reaction using irradiation. The photosensitive conjugation

based on click biochemistry can be carried out at mild con

ditions; independent of pH and temperature; Without affect

(51) Int, Cl,

Patent Application Publication

Dec. 22, 2011 Sheet 1 0f 21

Patent Application Publication

Dec. 22, 2011 Sheet 2 0f 21

Patent Application Publication

Dec. 22, 2011 Sheet 3 0f 21

Patent Application Publication

Dec. 22, 2011 Sheet 4 0f 21

Patent Application Publication

Dec. 22, 2011 Sheet 5 0f 21

Patent Application Publication

Dec. 22, 2011 Sheet 7 0f 21

Patent Application Publication

Dec. 22, 2011 Sheet 8 0f 21

Patent Application Publication

Dec. 22, 2011 Sheet 9 0f 21

rl|551 .52, 125.555

Patent Application Publication

Dec. 22, 2011 Sheet 10 0f 21

Patent Application Publication

Dec. 22, 2011 Sheet 11 0f 21

& Free llpase

Patent Application Publication

Dec. 22, 2011 Sheet 12 0f 21

Ma nnose Concentration (ppm)

Patent Application Publication

Dec. 22, 2011 Sheet 13 0f 21

lgM Concentration (ppm)

Patent Application Publication

Dec. 22, 2011 Sheet 14 0f 21

antitubulin antibody nanoparticles - <

antitubulin antibody nanopaiticles in

Patent Application Publication

Dec. 22, 2011 Sheet 15 0f 21

Patent Application Publication