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Laser Raman studies of interactions between antimicrotubular agents and cysteine
Journal of Molecular Structure, 143 (1986) 419-422 Elsevier Science Publishers B.V., Amsterdam -Printed
LASER RAMAN STUDIES
T. BOUCHAOUR1,
OF INTERACTIONS
S. TURRELLl,
.
'L.A.S.I.R.(L.P.2641 and Laboratoire 2 C.T.B.M.
C.N.R.S.),
I.N.S.E.R.M.
59045 Lille
(France)
BETWEEN
G. FLEURY',
de Physique,
419 in The Netherlands
ANTIMICROTUBULAR
M.-L. MALLEVAIS',
Universite
41nstitut
Chimie
and I. LESIEUR4
de Lille I, 59655 Villeneuve
Universiti? de Lille
U279, 59800 Lille
AGENTS AND CYSTEINE
59046 Lille
II,
(France)
'Unite
Pharmaceutique,
d'Ascq
(France)
I.N.S.E.R.M.
No. 16,
59045 Lille (France)
ABSTRACT
IR and Raman spectra have been obtained for a series of drug molecules which are known to be inhibitors of microtubular polymerisation. Raman spectra of drug-cysteine mixtures show distinct variations in the bands assigned to vC=C and vC=O of the drug and vSH of cysteine. Studies of drug-cysteine mixtures of varying concentrations suggest the formation of a 1:l complex. INTRODUCTION Syntheses methyl
in orye of our laboratories4
acrylophenones
have yielded
(ref.1):
x
-a
a group of 2-amino
CH2 II
0
C-C-CH2-Y d
where X=H(l) or C1(2), and Y= N
/
CH3 (A),
NCN-
\CH
In-vitro
3 tests have shown these compounds
bular polymerisation
with the inhibitory
(X=1, Y=C) > ILH (X=1, Y=A) > ILX (X=1,
to be powerful strength
inhibitors
decreasing
of microtu-
in the order
ILM
Y=B) >ILT (X=2, Y=B) > IL1 (X=2, Y=A).
ILY (ILX without the =CH2) is totally inactive (refs. 2 and 3).
The sum of bi-
ological
residue,
studies
the mechanism
We presented tures which
implies that these drugs interact
previously
indicated
communication
at a cysteine
but
is uncertain. (ref.4) spectroscopic
the formation
studies of ILH-cysteine
of a cysteine-drug
we report spectroscopic
sults of Raman studies of ILI-cysteine
results
complex.
mix-
In the present
for several drugs, as well as re-
mixtures
of varying concentrations.
METHODS The drugs were synthesized L-cysteine samples
was purchased
in KBr or polyethylene
0022s2860/86/$03.50
by methods
previously
from the Sigma Chemical
described
Co.
pellets were recorded
0 1986 Elsevier Science Publishers B.V.
(ref.l), while
IR spectra of powder on a Bruker FTIR IFS 113V
420 spectrometer IM water
over the range 4000-200
solutions
were recorded
using the 647.1 nm exciting teine an
from 4000-10
cm-l on a Coderg T800 spectrometer
line of a Coherent
Raman spectra were recorded
adjusted
Raman spectra of powder samples and
cm-l.
Radiation
of various k
BLg/
Krt laser. EysteinJ
Drug-cysratios with
pH of 1.
RESULTS AND DISCUSSION The Raman and IR frequencies presented
observed
for solid samples of five drugs are
in Table 1, along with proposed
assignments.
Our vibrational
TABLE 1 Infrared
and Raman frequencies
ILX Ramal 3055w 3007u 2975~ 2944~ 1665s 1623~ l595m
1414”
l304m 1256”
IR
1649s 1615~ 1590n 1569 1485m I4571 1415w I396ms I345 l314m
lO44m 1023~ I ooova 992~ 982m 960~
8“8V i87s 75 3w 718W 695m 6301, 615. 545w 5oom 475m 408v 36% 34 7v 335mw 2751~ 265~ 245~ 192ms
IR
3083” 3047w 3015mw 2980~ 2950v.br 1657s 1623” I5978
1659~ 1623~ l598m 1577 I467m 14481. l417m 1397ns I342ms
I304mw 1276s~ l244m ll98rs II66 l09lm 1068 1048 1019 997s
955m 931 899 837 ~~ 787s 754 711 692 632 522m 499 475 407
1153” ll34w
llama”
IR
3075.u
‘
l2l0mw 1204m llllw 1152”
ILEl
IL" R.m¶”
for some acrylophenone
I213mw I l83m ll55a II3610 I080
3025mw 2985ms 2962mw 1669~ 1624~ I5 96ms
1443vw
1659~s 1629” l598v 1578” 1466. 14490 1418. 1397. 1349m
I305w
1302~
1243~
1246,x 1213~ I l83m
1200” ll78mw ll58w
I
ILT R.ma”
10001 992m
307ov
2991~ 2962~ 1644~~ ,620~ 1587ma
*Mm 817u
97lv
037 817
868~ 82Ov 803nw
757v 7201~ 680~
761s 719ms 700ma
618,~
620 560
5oov 41% 4oov 385v 297~ 270~
1666s 1626~ 1597s 1576 1469” 1446~~s 1423~ 1423~ 1371 I348 l318m 1290~ 126611
1199s
l207m 1175”
IIZIS 1080. 1063 1052 1014 999s
ll38v 1089,
614~ .^^
761s 72001 7Olm 678 620 559
887vv 866m 785m 753ms 650x, 615m 590v,br
89lm 840m
755s 718 6968
408w
477mv 412
460 44ow 363~
235~
1207,~ ll7Ou 11551ar ll35m 1092ms
lOl3mu 999w 985 970” 955” 930m 85Oma 793s 750m 715w 6J5m 629~
436
510w.br 455m 407v 385v 353”
228s 1800s
I IOm, 85s
1423~ 1400n.br I335m 1290~ 1290~
793v 752s 715v 675~ 629m
262. 243~ l97w 174” 137s lllm
CH2
“C-O “C-C
147ou
840s
33Ou 273~
277~
1655ms 1623~ I5850
620
5Y”W
465~ 435
”
VCH2
VCH. “CH; T sens sell*
0 6CH 6CH2 T sell* 6CH2 ucH2
1015w IOOOU
957u 923
7l8mr
VW* II II
.I
I56m ll36m 108lv
837m.d 817
Assignments IR
x IZOOW II75
lOlZ*
977ms 956 922m
Raman
302Ov.br 2975”. 2944~ 1655s 1623” 1589s
1060~~ I028 1016 993ms
ILI IR
3072u
1420~ I389w
derivatives.
18Od
552~ 512~
+ ‘ICH TCH ICH Y sens PC"2 0 -Y YC” Y ring Wli 6$ 64
spectra
421 and assignments
for L-cysteine
note that unlike
the frequency
series of compounds, A cysteine-drug
vC=C which remains
the frequency
by Susi (ref.5).
virtually
uC=O is strongly
constant
dependant
for the identification
for the
the sulfur or ni-
and the C=O or C=C groups of the drug.
regions of most importance
Hence, the
of such a bond would
be a) for cysteine:
vSH, uCS and vNH3+, which are observed
cm -l, respectively.
b) for the drug: vC=C, vC=O and v$, which are observed
1623, 1655-1677
at
2575, 690 and 3064
and 1585-1599cm-', respectively.
In Fig. 1 we present
Fig. 1. Raman spectra
these spectral
of ILI-cysteine
regions
for ILI-cysteine
mixtures.
We
on X and Y.
bond would most likely occur between either
trogen groups of cysteine spectral
agree with those published
mixtures.
at
422
When R=l, the vNH3+ region remains unchanged in going from free cysteine to the mixture, but the vSH and vCS bands virtually disappear. In addition, while v$ for the drug remains unchanged, the band due to vC=C nearly disappears and the frequency of vC=O shifts from 1655 to 1675 cm-l ( the same frequency as that observed for vC=O in ILY), thus implying a decrease in conjugation. In order to test the percentage of drug molecules necessary for total complexation with cysteine, we examined Raman spectra of solutions for which R= 2, 1 and 0.5. When R=2 we observe indications of intermediate stages of interaction. In particular there are two vC=O frequencies due to the simultaneous presence of free and complexed ILI. No appreciable spectral changes seem to occur between R=l and R=0.5. Our observations are consistant with a model by which the cysteine sulfur binds at the C=C site of the drug. Moreover, the complexation appears to be stable and complete when R -<1. REFERENCES 1 M. Cazin, et al., Biochemical Pharmacology, (1984) (In press). 2 I. Lesieur, et al., Arzneimittel Forth.. (1984) (In press). 3 A. Delacourte, et al., Meth. and Find. Exptl. Clin. Pharmacol. (1984) (In press). 4 T. Bouchaour, et al., Proc. I Europ. Conf. on Spec. of Biol. Mol., (1985)
.
J. Mol. Structure, 102 (1983) 63-78.
LASER RAMAN STUDIES
T. BOUCHAOUR1,
OF INTERACTIONS
S. TURRELLl,
.
'L.A.S.I.R.(L.P.2641 and Laboratoire 2 C.T.B.M.
C.N.R.S.),
I.N.S.E.R.M.
59045 Lille
(France)
BETWEEN
G. FLEURY',
de Physique,
419 in The Netherlands
ANTIMICROTUBULAR
M.-L. MALLEVAIS',
Universite
41nstitut
Chimie
and I. LESIEUR4
de Lille I, 59655 Villeneuve
Universiti? de Lille
U279, 59800 Lille
AGENTS AND CYSTEINE
59046 Lille
II,
(France)
'Unite
Pharmaceutique,
d'Ascq
(France)
I.N.S.E.R.M.
No. 16,
59045 Lille (France)
ABSTRACT
IR and Raman spectra have been obtained for a series of drug molecules which are known to be inhibitors of microtubular polymerisation. Raman spectra of drug-cysteine mixtures show distinct variations in the bands assigned to vC=C and vC=O of the drug and vSH of cysteine. Studies of drug-cysteine mixtures of varying concentrations suggest the formation of a 1:l complex. INTRODUCTION Syntheses methyl
in orye of our laboratories4
acrylophenones
have yielded
(ref.1):
x
-a
a group of 2-amino
CH2 II
0
C-C-CH2-Y d
where X=H(l) or C1(2), and Y= N
/
CH3 (A),
NCN-
\CH
In-vitro
3 tests have shown these compounds
bular polymerisation
with the inhibitory
(X=1, Y=C) > ILH (X=1, Y=A) > ILX (X=1,
to be powerful strength
inhibitors
decreasing
of microtu-
in the order
ILM
Y=B) >ILT (X=2, Y=B) > IL1 (X=2, Y=A).
ILY (ILX without the =CH2) is totally inactive (refs. 2 and 3).
The sum of bi-
ological
residue,
studies
the mechanism
We presented tures which
implies that these drugs interact
previously
indicated
communication
at a cysteine
but
is uncertain. (ref.4) spectroscopic
the formation
studies of ILH-cysteine
of a cysteine-drug
we report spectroscopic
sults of Raman studies of ILI-cysteine
results
complex.
mix-
In the present
for several drugs, as well as re-
mixtures
of varying concentrations.
METHODS The drugs were synthesized L-cysteine samples
was purchased
in KBr or polyethylene
0022s2860/86/$03.50
by methods
previously
from the Sigma Chemical
described
Co.
pellets were recorded
0 1986 Elsevier Science Publishers B.V.
(ref.l), while
IR spectra of powder on a Bruker FTIR IFS 113V
420 spectrometer IM water
over the range 4000-200
solutions
were recorded
using the 647.1 nm exciting teine an
from 4000-10
cm-l on a Coderg T800 spectrometer
line of a Coherent
Raman spectra were recorded
adjusted
Raman spectra of powder samples and
cm-l.
Radiation
of various k
BLg/
Krt laser. EysteinJ
Drug-cysratios with
pH of 1.
RESULTS AND DISCUSSION The Raman and IR frequencies presented
observed
for solid samples of five drugs are
in Table 1, along with proposed
assignments.
Our vibrational
TABLE 1 Infrared
and Raman frequencies
ILX Ramal 3055w 3007u 2975~ 2944~ 1665s 1623~ l595m
1414”
l304m 1256”
IR
1649s 1615~ 1590n 1569 1485m I4571 1415w I396ms I345 l314m
lO44m 1023~ I ooova 992~ 982m 960~
8“8V i87s 75 3w 718W 695m 6301, 615. 545w 5oom 475m 408v 36% 34 7v 335mw 2751~ 265~ 245~ 192ms
IR
3083” 3047w 3015mw 2980~ 2950v.br 1657s 1623” I5978
1659~ 1623~ l598m 1577 I467m 14481. l417m 1397ns I342ms
I304mw 1276s~ l244m ll98rs II66 l09lm 1068 1048 1019 997s
955m 931 899 837 ~~ 787s 754 711 692 632 522m 499 475 407
1153” ll34w
llama”
IR
3075.u
‘
l2l0mw 1204m llllw 1152”
ILEl
IL" R.m¶”
for some acrylophenone
I213mw I l83m ll55a II3610 I080
3025mw 2985ms 2962mw 1669~ 1624~ I5 96ms
1443vw
1659~s 1629” l598v 1578” 1466. 14490 1418. 1397. 1349m
I305w
1302~
1243~
1246,x 1213~ I l83m
1200” ll78mw ll58w
I
ILT R.ma”
10001 992m
307ov
2991~ 2962~ 1644~~ ,620~ 1587ma
*Mm 817u
97lv
037 817
868~ 82Ov 803nw
757v 7201~ 680~
761s 719ms 700ma
618,~
620 560
5oov 41% 4oov 385v 297~ 270~
1666s 1626~ 1597s 1576 1469” 1446~~s 1423~ 1423~ 1371 I348 l318m 1290~ 126611
1199s
l207m 1175”
IIZIS 1080. 1063 1052 1014 999s
ll38v 1089,
614~ .^^
761s 72001 7Olm 678 620 559
887vv 866m 785m 753ms 650x, 615m 590v,br
89lm 840m
755s 718 6968
408w
477mv 412
460 44ow 363~
235~
1207,~ ll7Ou 11551ar ll35m 1092ms
lOl3mu 999w 985 970” 955” 930m 85Oma 793s 750m 715w 6J5m 629~
436
510w.br 455m 407v 385v 353”
228s 1800s
I IOm, 85s
1423~ 1400n.br I335m 1290~ 1290~
793v 752s 715v 675~ 629m
262. 243~ l97w 174” 137s lllm
CH2
“C-O “C-C
147ou
840s
33Ou 273~
277~
1655ms 1623~ I5850
620
5Y”W
465~ 435
”
VCH2
VCH. “CH; T sens sell*
0 6CH 6CH2 T sell* 6CH2 ucH2
1015w IOOOU
957u 923
7l8mr
VW* II II
.I
I56m ll36m 108lv
837m.d 817
Assignments IR
x IZOOW II75
lOlZ*
977ms 956 922m
Raman
302Ov.br 2975”. 2944~ 1655s 1623” 1589s
1060~~ I028 1016 993ms
ILI IR
3072u
1420~ I389w
derivatives.
18Od
552~ 512~
+ ‘ICH TCH ICH Y sens PC"2 0 -Y YC” Y ring Wli 6$ 64
spectra
421 and assignments
for L-cysteine
note that unlike
the frequency
series of compounds, A cysteine-drug
vC=C which remains
the frequency
by Susi (ref.5).
virtually
uC=O is strongly
constant
dependant
for the identification
for the
the sulfur or ni-
and the C=O or C=C groups of the drug.
regions of most importance
Hence, the
of such a bond would
be a) for cysteine:
vSH, uCS and vNH3+, which are observed
cm -l, respectively.
b) for the drug: vC=C, vC=O and v$, which are observed
1623, 1655-1677
at
2575, 690 and 3064
and 1585-1599cm-', respectively.
In Fig. 1 we present
Fig. 1. Raman spectra
these spectral
of ILI-cysteine
regions
for ILI-cysteine
mixtures.
We
on X and Y.
bond would most likely occur between either
trogen groups of cysteine spectral
agree with those published
mixtures.
at
422
When R=l, the vNH3+ region remains unchanged in going from free cysteine to the mixture, but the vSH and vCS bands virtually disappear. In addition, while v$ for the drug remains unchanged, the band due to vC=C nearly disappears and the frequency of vC=O shifts from 1655 to 1675 cm-l ( the same frequency as that observed for vC=O in ILY), thus implying a decrease in conjugation. In order to test the percentage of drug molecules necessary for total complexation with cysteine, we examined Raman spectra of solutions for which R= 2, 1 and 0.5. When R=2 we observe indications of intermediate stages of interaction. In particular there are two vC=O frequencies due to the simultaneous presence of free and complexed ILI. No appreciable spectral changes seem to occur between R=l and R=0.5. Our observations are consistant with a model by which the cysteine sulfur binds at the C=C site of the drug. Moreover, the complexation appears to be stable and complete when R -<1. REFERENCES 1 M. Cazin, et al., Biochemical Pharmacology, (1984) (In press). 2 I. Lesieur, et al., Arzneimittel Forth.. (1984) (In press). 3 A. Delacourte, et al., Meth. and Find. Exptl. Clin. Pharmacol. (1984) (In press). 4 T. Bouchaour, et al., Proc. I Europ. Conf. on Spec. of Biol. Mol., (1985)
.
J. Mol. Structure, 102 (1983) 63-78.