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Interaction of cis-pt(ino)2Cl2 with amino acids
Interaction of ck-Pt(ino)2C12 with Amino Acids A. Garoufis, R. Haran, M. Pasdeloup, J. P. Laussac, and N. Hadjiliadis AG, NH. Department of Chemistry, University of Ioannina, Greece.-RH, Laboratoire de Chimie de Coordination du CNRS, Toulouse, France
MP, JPL.
ABSTRACT The reactions of ci.s-Pt(ino)2C12 with the amino acids Gly, L-Ala, L-Val, L-Ileu, L-Phe and L-Pro were studied in methanolic solutions. The (1: 1) adducts of the formulas cis-[Pt(ino)z(am-ac)]C1 were isolated from these reactions in the solid state, which in turn produce the cis-[Pt(ino)z(am-acH)C1]C1 complexes, by treating the former with equivalent amounts of HCl, in aqueous solutions. The complexes were characterized with elemental analysis, conductivity measurements, IR, ‘H NMR, and 13C NMR spectra. The results show that the purine ring of inosine interacts with the aliphatic side chain of the amino acids. The platination increases the percentage of the C3 f -endo-anti conformation of the sugar part of inosine.
INTRODUCTION Nucleic acids and proteins can recognize each other selectively in biological systems and interact through specific amino acid side-chains and nucleic acid bases [ 11. These specific interactions can in several cases be mediated through a metal ion, helping in the formation of ternary complexes [2, 31. For example, Cu+ z and Zn+’ ions can provoke interactions between polypeptides containing tyrosine and glutamic acid fragments and polynucleotides [4]. The various ligand-ligand interactions may be classified as (i) steric hindrance, (ii) electrostatic interactions and hydrogen bonds, (iii) hydrophobic interactions, and (iv) metal ion mediated electronic interactions [5]. Sigel et al. [l] studied the ternary complexes formed between ATP, the metal ions Mn+2, CU+~, Zn+2, Cd+2, and Pbf2 and various amino acids with increasing aliphatic
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Department
Journal of Inorganic Biochemistry 31, 65-79 (1987) @ 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
of Chemistry,
65 0162-0134/87/$3.50
66 A. Garoufis et al.
side-chains. They found that hydrophobic interactions existed between the aliphatic side chains of the amino acids and the aromatic purine ring of ATP, which increased with the length of the chain. Similar studies between various peptides and nucleotides have also been carried out by Martin et al. [6-81, who observed similar ligand-ligand interactions. Yamauchi et al. [5, 9-121 also found similar ligand-ligand interactions in mixed amino acid-metal complexes. The same is true for the recently published PdGHL-5 ‘-IMP and 5 ‘-GMP ternary system [13], where, based on the ‘H NMR spectra, a very strong interaction between the imidazole ring of the peptide and the purine ring of the bases, was observed. In the present paper we describe the interaction of cis-Pt(ino)2Clz, a compound of cis geometry with possible antitumor activity [14], and various amino acids in methanolic solutions. This is part of a more general project of ternary complexes of Pt +2 or Pd+2, nucleosides or nucleotides, and amino acids or peptides undertaken by us in order to detect and characterize such ligand-ligand interactions. In addition, such studies are also interesting because of the anticancer properties of cis-DDP. Despite the fact that cis-DDP interacts with DNA during its replication, thereby demonstrating its anticancer properties [ 15-171, its interaction with the other chemical constituents of the body is not excluded and has not yet been elucidated [ 181. For example, cis-DDP reacts with certain small molecules of the blood plasma, producing complexes of low molecular weight, essential for the availability and the elimination of Pt(I1) from the organism [ 191, according to the reaction Protein-Pt
* complex-t
Protein + Pt-small
molecules
(1)
Finally, the new mixed complexes of Pt + 2 with amino acids and nucleosides having the cis configuration might also be potential anticancer agents, as in other similar cases [20-221. Several of the complexes are currently under screening against various tumors.
RESULTS
AND DISCUSSION
The amino acids glycine, L-alanine, L-valine, L-isoleucine, t_-proline, and Lphenylalanine, in their sodium salt forms, were used in this study to prepare 1: 1, N,O chelate complexes with cis-Pt(ino)2C12 [23], in methanolic solutions, according to cis-Pt(ino)2C12 + am - ac - Na CHZoHcis-[Pt(ino)s(am-ac)]Cl + 0 II The Pt-O-Cbond of the amino acids, in these complexes, in 0.1 N HCl solution, without breaking of the Pt-NH2-bond: cis-[Pt(ino)2(am-ac)]C1
NaCl
(2)
could be easily broken
“z’ cis-[Pt(ino)2(am-acH)C1]C1
(2)
The elemental analyses of the 12 complexes obtained in this way agree with their empirical formulas and are given in Table 1. The complexes of both series are 1: 1 electrolytes as expected, in aqueous solutions, and the values of the molar conductivities are also included in Table 1. The structures and the conformation of the complexes in the above series are mainly deduced from the NMR and IR spectra, which are briefly discussed below.
INTERACTIONS OF cis-Pt(ino)rC& WITH AMINO ACIDS
TABLE 1. Elemental
Analysis and Conductivity
Complexes
cis-[Pt(ino)s(Gly)]Cl
cis-[Pt(ino),(Ileu)]Cl
cis-[Pt(ino),(Phe)]Cl
ck-[Pt(ino),(GlyH)Cl]Cl
cis-[Pt(ino),(AlaH)Cl]Cl
cti-[Pt(ino),(ValH)Cl]Cl
cis-[Pt(ino)#roH)ClJCl
ci.s-[Pt(ino),(Ileu)Cl]Cl
Pt(%)
67
Values of the Complexes
Cl(%)
C(W)
H(%)
N(%)
Calc
23.19
4.21
31.41
3.33
14.98
Found
23.03
3.87
31.40
3.50
14.70
Calc
22.81
4.14
32.28
3.51
14.73
Found
22.40
3.80
31.92
3.22
14.65
WC.
22.09
4.01
33.97
3.85
14.27
Found
21.85
3.65
33.61
3.98
14.20
Calc.
22.14
4.02
34.05
3.63
14.30
Found
21.15
4.20
33.90
3.85
14.21
Calc.
21.74
3.95
34.78
4.01
14.04
Found
21.41
4.32
34.65
4.25
13.85
talc.
20.95
3.80
37.31
3.65
13.53
Found
20.58
4.16
36.98
3.53
13.75
Calc.
22.25
8.09
30.12
3.31
14.37
Found
23.00
8.16
29.81
3.45
14.15
talc.
21.90
7.96
30.99
3.48
14.15
Found
21.83
8.51
30.85
3.58
14.27
talc.
21.23
7.72
32.66
3.81
13.72
Found
21.91
7.95
32.35
4.05
13.62
Calc.
21.28
7.73
32.73
3.60
13.75
Found
21.41
7.91
32.55
3.48
13.59
Calc.
20.92
7.60
33.45
3.91
13.51
Found
21.25
7.42
33.15
4.15
13.32
mole-‘)
116
113
101
105
103
123
90
98
98
93
97
rH NMR and 13C NMR Spectra The spectra were recorded in DrO solutions and the chemical shifts are given in Table 2. The N7 coordination of inosine in cis-Pt(ino)$& [ 141 is retained in the complexes of both series, since the Hs proton is always shifted downfield more than the Hz proton and 0.7-0.9 ppm, as compared to the free ligand. The chemical shifts of the amino acid protons in the complexes are very small compared to the free ones. Sigel et al. [2] observed upfield shifts of the amino acid protons, with aliphatic side-chains, in ternary complexes with ATP, and explained the phenomenon as due to ligand-ligand hydrophobic interactions. These interactions were stronger as the size of the aliphatic side-chains of the amino acids was increased. In our ternary systems, the chemical shifts of the amino acid protons are the average of downfield shifts due to Pt(II) coordination and upfield shifts due to hydrophobic
8.055
8.145
8.152
8.165
8.830
8.891
8.902
8.912
cis-[Pt(ino),(Gly)Cl]Cl
cis-[Pt(ino),(Val)Cl]Cl
cis-[Pt(ino),(Ileu)Cl]CI
6.106
6.095
6.087
6.048
6.043 6.049
6.003 6.086 6.060
4.624
4.616
4.604
4.622
4.611 4.619
4.688 4.604 4.603
4.417
4.408
4.399
4.400
4.391 4.400
4.376 4.396 4.392
4.248
4.249
4.252
4.238
4.227 4.239
4.217 4.249 4.230
3.962
3.954
3.945
3.927
3.920 3.931
3.869 3.942 3.930
3.852
3.845
3.836
3.815
3.808 3.814
3.779 3.833 3.821
1.039 AS = + 0.014 0.986 1.014 AS = + 0.020 0.938 = +0.028
0.975 A6 = + 0.003 1.025 0.990 A.6= i-0004 0.910 A&= -0.008
1.479 A6 = + 0.029
3.574 A6 = + 0.047
1.456 AIS= +0.006
3.539 A6= +0.012
CHz
3.610 A6 = + 0.024 3.679 Ah= +0.043
2.010 A.6= +0.060
3.651 A&= +0.015
3.59 A6 = + 0.004
CH,
2.275 Ad = + 0.030
1.955 A&= +0.005
2.25 AS = + 0.005
CHo
Amino acid protons
Academic, New York, 1971. The positive shift signifies down field shift and the negative one uptield shift relative to the free amino acids.
in chemical shift from the positions of the free amino acids, taken from A. F. Casy, PMR Spectroscopy in Medicinal and Biological Chemislry,
8.055 8.055
8.833 8.830
cis-[Pt(ino),(Pro)]Cl cis-[Pt(ino)2(Val)]C1
u AS. The difference
8.123 8.144 8.089
8.251 8.888 8.855
Inosine cis-Pt(ino)zC12 cis-[Pt(ino),(Gly)]Cl
Complexes
in ppma ~._____
Inosine protons ___.__
TABLE 2. ‘H NMR Chemical Shifts of the Complexes
INTERACTIONS
OF cis-Pt(ino)rQ
WITH AMINO
ACIDS
69
interaction of the side-chain of the amino acids with the aromatic purine rings of the two inosine molecules. The result is a very small downfield shift (0.003-0.015 ppm) in most cases in the chelated complexes cis-[Pt(ino)(am-ac)]Cl. An upfield shift of 0.008 ppm is observed only in the case of the complex with Ileu, in one of its -CH3 resonances. The existence of a synergic effect of upfield and downfield shifts is obvious in the present system, since the downfield shifts due to coordination to Pt(I1) are much larger in binary Pt(II)-amino acid complexes, ranging from 0.2 to 0.8 ppm [24]. This implies a hydrophobic ligand-ligand interaction. This interaction is even larger in the chelated N,O than in the nonchelated series, since the downfield chemical shifts are larger in the latter. This might be the result of a more fixed position in space of the aliphatic side-chains in the former series, bringing it closer to the purine rings and hence to a stronger interaction. In the nonchelated species, on the other hand, this group can be a little further from the purines as subject to free rotation of the amino acid around the Pt-NH2 bond. The ligand-ligand hydrophobic interaction is also shown from the upfield shift by 0.05 ppm of the Hs and 0.089 ppm of the H2 proton of inosine, in the ternary N,O chelated systems, with L-proline, L-valine, and t_-isoleucine. In the second series of complexes, on the other hand, very slight downfield shifts of 0.003-0.024 ppm for the Ha protons and of 0.001-0.021 ppm for the H2 protons are observed. Upfield shifts are also observed for the Hi g protons of the sugar part of inosine, in the first series of complexes by 0.026-0.042 ppm and in the second downfield shifts by 0.001-0.020 ppm. Strong ligand-ligand interaction was also observed in the GHL-Pd(II)nucleotide system [13], though between aromatic rings. The t3C NMR spectra of the complexes cis-[Pt(ino)2(Ileu)]C1 and cis-[Pt(ino)z(IleuH)C1]C1 were recorded in D20 solutions and confirm the N7 coordination of inosine in both compounds, the N,O chelation of Ileu in the first, and the N coordination of Ileu in the second. Thus, the Ca near the coordination site N7 of inosine shifts downfield in the first complex by 4.16 ppm and by 5.56 ppm in the second, while the C5 atom shifts upfield by 1.42 ppm in the first and by 1.00 ppm in the second. The shifts of the other carbons are given in Table 3. Although the carbonyl carbon resonance is not seen in the I3C NMR spectra of both complexes, the C, of Ileu (near the -COOgroup) shifts by 1.60 ppm in the complex cis-[Pt(ino)z(Ileu)Cl]Cl and by 1.92 ppm in the cis-[Pt(ino)2(Ileu)]Cl, 0 II indicating Pt-O-Cbonding in the latter complex. The influence of Pt(II)-inosine and amino acid coordination to the conformation of the sugar moiety of the nucleoside was further explored, by measuring the coupling constants of the various sugar protons in the ‘H NMR spectra [25-331. The furanose ring of the sugar in many nucleosides is in equilibrium between the Cj’-endo, anti = 3E and the C2 I -endo, anti = z E conformations (Figure la) and metal coordination to the bases shifts the equilibrium towards the former [25-331. The conformation around the C(4’)-C(5’) bond, on the other hand, described as gg, gt or tg [34-371, was found not to favor the gg conformer on passing from G5 ‘p to its complex with K,PtCI, [29], while it is favored in its complex with cis- and transPt(NH3)$12 [26-281 (Figure lb). A method for the calculation of the various sugar conformers of nucleosides, based on the ‘H NMR spectra, was first proposed by Sarma et al. [34-371 and is based on the estimation of the coupling constants 3J nn and application of the Karplus equation. The
-
157.66
-
160.40
c6
-
149.19
-
151.88
c2
-~-
-
146.93
150.06
150.06
c4
-
139.89
145.45
144.05
c8
CS
-
125.32
124.32
123.90
__~_______~
Purine
Inosine
-
88.73
92.00
91.87
C,f
-
86.62
87.91
88.03
C4’
with Ileu, in ppm
-
75.13
77.18
76.98
CZ’
Sugar
-
11.22
72.30
72.49
Cjf
-
62.24
63.39
63.58
cg’
._ -_--.-_-
60.30
-
61.90
62.22
c,
36.50
-
38.43
38.50
c,
-.-.-.----.--_-
a Taken from A. J. Jones, D. M. Grant, M. W. Winkley, and R. K. Robins, J. Am. Chem. Sot. 92, 4079 (1970). b Taken from A. F. Casy, PMR Spectroscopy in Medicinal nnd Biological Chemistry, Academic, New York, 1971.
Isoleucineh
Inosine”
cti-[Pt(ino)2(Ileu)C1]C1
cis-[n(ino),(Iieu)]Cl
Compounds
_-
TABLE 3. 13C NMR Chemical Shifts of the Complexes
25.10
-
27.05
27.05
C”
15.30
-
17.16
17.29
c,
Amino acid G
11.70
-
13.65
13.65
--
174.60
-
-
co
INTERACTIONS
OF cis-Pt(ino)&
573
C&end0
WITH AMINO ACIDS
71
C&end0
a
“4, QQ
HO
<:a,
HO
“5‘
H4’ Ql
b FIGURE_ 1. The C3 f-end0 and the Cz I -endo sugar conformations, with 0 carbon and 0 oxygen atoms. (b) The sugar conformation around the C4t-C5 1 bond.
calculation of the gg, gt, or tg conformers around the C(4 ‘ )-C(5 ’ ) bond has also been given by Sarma [34]. The results for inosine, cis-Pt(ino)Q, and the various complexes of the type cis-[Pt(ino)2(am-ac)]Cl and cis-[Pt(ino)z(am-acH)Cl]Cl are summarized in Table 4, and typical spectra are shown in Figure 2. It is seen that the complexation of K$tC& to inosine favors the C3j endo anti conformation in the cis-Pt(ino)&& complex, increasing it to 59 % from 46% in the free ligand. The gg percentage is also favored in the complexes ranging from 71% to 80%, but not differing appreciably from the free ligand (74%). The amino acid complexations in the N,O chelated forms, do not affect the Kq = %3E/%2E significantly (Table 4). A slight decrease in the %3E is, however, observed in the glycine and proline complexes, while in the valine and isoleucine derivatives there is a further decrease of the %3 E endo, being 50% in both. This reflects the greater size of the amino acid aliphatic side-chains, producing larger interactions with the purine rings. It increases slightly again to 55 % in the nonchelated species. For the percent of the gg conformers the opposite effect is observed, e.g., slight decrease in the nonchelated complexes (See Table 4). It is worthwhile mentioning that the sugar pucker of inosine in the solid state is in the C3’ endo anti gt conformation [38]. Thewalt et al [39] found two independent inosine molecules in its crystal structure, the one assuming the CZ~ endo anti gg conformation and the other the C3/-endo anti gg conformation). The S/-IMP disodium salt on the other hand, has the C(2 ‘) endo anti gg conformation in the solid state [40]. The conformation of the sugar in the disodium salt of 5 ‘-IMP does not change in the crystal structure of a nonstoichiometric compound of cis-Pt(NHW12, being again the C(2 ‘) endo anti gg [41]. In a Cu(II) complex of 5 ‘-IMP disodium salt and dien, the same pucker of the sugar was once more found [42], while’ in a similar one with 2,2’-bipyridyl the C3 I endo anti gg conformation predominates [43]. Finally the t and g + h conformations around the R
I -CH,-CH,-
INTERACTIONS
5,O
8.0 FIGURE
OF cis-Pt(ino)*Q
2.
4,o
WITH AMINO
P P r1
ACIDS
73
281)
The 250 MHz, ‘H NMR spectrum of the complex cis-[Pt(ino)z(val)]C1.
bond of L-Val and L-Ileu were calculated, in the complexes with these two amino acids, from the values of the coupling constants JHaHBgiven in Table 5 and using JT = 13.3 Hz and JG = 2.4 Hz [6-81. The calculation of the individual h and g conformers is not possible in this case, since the amino acids do not possess a CH,,,-CH Z(ojsystem. The results are included in Table 5 and show an about 85 %-I36 % abundance of the h + g conformers for the Ileu complexes and 82%-88 % for the L-Val complexes. In similar Pd(I1) complexes with dipeptides containing L-Val or L-Ileu, the h + g conformers were predominant (90%) [8]. The same was true in similar mixed Pd(II)-depeptide nucleotide complexes [6]. In the GHL-Pd-nucleotide complex, on the other hand, the h conformer was more than 80 % [ 131. The increase of the percentage of h + g conformers in mixed Pd(II)-amino acid complexes has been interpreted as due to increase of the ligand-ligand hydrophobic interactions in these systems [9, 121. Thus the higher values of this sum in the complexes with L-Ileu than with L-Val may show a stronger such interaction in the first ones, as expected. IR Spectra In the region below 1800 cm-‘, several bands can be identified and help to better understand the structures of the complexes. The characteristic IR frequencies are given in Table 6. In the present study, many bands mainly due to the various -NH*, and -CH’motions of the amino acids were identified as follows: Glycine in its zwitterionic form, shows the v,(COO-) at 1610 cm-’ and the &(NH3+) at 1585 cm-’ [44,45]. In the complex TABLE 5.
‘H NMR Coupling Constants and Conformations around the -CH -CHR( bonds (x)(a) (B) 0)
g+h Complexes ci.+[Pt(ino)~(Val)]Cl cis-[Pt(ino),(ValH)Cl]Cl cis-[pt(ino)z(Ileu)]Cl cis-[F’t(ino)z(IleuH)C1]C1
JBX
4.359 4.325 3.975 3.902
(%o)
18 17 15 14
82 83 85 86
cis-lFY(ino),(Val)lCi
Ino
Complexes
c,
1600s
1396s
-
_
-
_
_
1625s
1330~1 1620s
_
163&h
541m
-
540m
113h
1118s
1128m
804m
804m
804m
1298m
1308m
_
_
_
1342s
160&h
1459m
-
1595s 156Om 1515s
1700s
1310s
1335m
1362s
1432m
VI’-Cl
1595s 156Om 1520~1
1600s
1705s
_
Gff-NCCO
_
16Ctsh
17Oas -
6(NHz) p,(NHz) pw(NHz) pw(CQO-)
1407s
6(CH)
1595s 156Om 152OW
&CHz) p&H,)
334w
S(CH3)
1591s 1555s 1519m
v,(CHJ
_.
1698s
v@mc-) amino acid
1592 155lm 1518111
C=N) ring
v(c =
1690s
acid
amino
v.(coo-)
IR Bands of the Complexes
u.(C=O) skeletal
TABLE 6. Characteristic
831m
832111
830m
828m
822m
INTERACTIONS
E
Fi OD 3
5 I
I
I I
x z I I
I
I
I
I
OF cis-Pt(ino)zClz
WITH AMINO ACIDS
75
76 A. Garoufis et al.
cis-[Pt(ino)(Gly)]Cl, the broad band at 1630 cm-’ is assigned to the overlapping of the coordinated to Pt(II), 6(NH2), and v,(COO-) of the amino acid [23]. The intensity of this band diminishes upon deuteration and a new weak band appears at - 1600 cm- ‘, obviously due to the metal coordinated v,(COO-) mode. On passing to the cis[Pt(ino)z(GlyH)C1]C1 complex, a band of medium intensity appears at 1634 cm-‘, which disappears upon deuteration, due to the metal coordinated &NH>) motion. On the other hand, the intensity of the band at 1700 cm- ’ due to the VC = 0 of the keto group at the sixth position of inosine, is enhanced, since it coincides now with the v,(COOH) group [23]. The same behavior is also observed in all the other complexes, confirming the N,O chelation of the amino acids in the first series and their N coordination in the second. In a few cases the &NH*) and the v,(COO-) of the metal coordinated amino acids are shown as two different bands, Such is the case with L-ala, in the complex cis[Pt(ino)2(ala)]C1 showing the 6(NH2) at 1629 cm-. ’ and the v,(COO-) at 1597 cm- ‘. The metal coordinated v,(COO-) of the complex cis-[Pt(ino),(Gly)]CI is assigned to a new band at 1407 cm-~ I, while it occurs at 1412 cm- I in free Gly [44]. It is shown at 1362 cm- 1in the complex with L-ala, possibly coupled with other motions. It occurs at 1374 cm-’ in the Pt(L-ala)z complex [45]. It is also seen at 1342 cm--’ in the corresponding complex with t_-val [46]. at 1338 cm-’ in the complex with L-pro, and 1383 cm-’ in the complex with L-Ileu. All these bands disappear in the second series of complexes indicating again the existence of free -COOH groups in them. The A(Y, - YJ of the -COOgroup varies from 223 to 267 cm ’ in the various Pt(I1) complexes, in agreement with other similar complexes [45, 461. The 6(-CH) and p(-CH) of the various aliphatic groups of the amino acids can be identified in the region of 1250- 1450 cm - I. These bands shift only slightly in the two series of complexes, compared to the free amino acids, since they are rather far from the coordination sites. More such bands are identified in the complexes with L-Val and L-Ileu, containing more aliphatic side-chains (See Table 6). A band near 1300 cm -~’ appearing constantly in all the complexes of the first series and disappearing in the second, should be associated with a deformation motion of the chelated five-membered , N-C
I ’ o-c
Pt
ring. The same is true with the band at 804 cm- I, which disappears in the second series of complexes and which may be assigned to the p,(COO- ) [45, 461. A band near 540 cm- ’ that appears in the complexes of both series disappears upon deuteration and is assigned to the metal coordinated &NH,) motion. A medium intensity band near 330 cm- ’ appears in the second series of complexes cis-[Pt(ino),(am-acH)Cl]Cl and is assigned to the vPt-Cl, since it is absent in the first series of complexes, with N,O chelated amino acids cis-[Pt(ino)2(am-ac)]Cl. Finally, inosine shows a band at 822 cm-‘, which shifts to 828 cm ’ in the complex cis-Pt(ino)$l;! and the second series of complexes and at 830 cm ’ in the first series. This has been assigned to a sugar-ring vibrational mode and might be related to the increase of the percentage of the C 3j-e&o-anti conformer of inosine, on passing to its Pt(I1) complexes [28. 29, 331. In the case, however, of guanosine. the opposite effect was observed, e.g., removal of the 824 cm- I of the free ligand, with a higher
INTERACTIONS
OF cis-Pt(ino)Q
WITH AMINO
ACIDS
77
percentage of the CZ I-endo-anti conformer, to 797-798 cm-’ in its Pt(I1) complexes with increased proportion of the C3 ‘-endo-anti conformer [28, 29, 331. EXPERIMENTAL Materials Glycine, L-Alanine, L-Valine, L-Isoleucine, L-Proline, L-Phenylalanine, and Inosine were purchased from Aldrich Chemical Company and Fluka A.G. Potassium tetrachloroplatinate was a gift of Degussa AG (West Germany). Methods (i) The elemental analyses of Pt and Cl were performed in the laboratory of Inorganic Chemistry at Ioannina, while those of C, H, and N were performed in the Laboratoire de Chimie de Coordination, in Toulouse. (ii) The conductivity measurements were performed in an E365B Conductoscope, Metrohm Ltd., Herisau, Switzerland. (iii) The IR spectra were recorded on a Perkin Elmer model 580 spectrophotometer. (iv) The ‘H NMR spectra were obtained on a Brucker WM-250 spectrometer equipped with an Aspect 3000 computer. (v) 13C NMR spectra were recorded at 22.63 MHz on a Brucker WH-90-NMR spectrometer, operating in the pulse Fourier transform mode. Both ’ H and 13C chemical shifts were measured in parts per million with DSS as reference. Preparation
of the Compounds
The starting material cis-Pt(ino)&lz was prepared from K,PtCI, and inosine, according to a published method [ 141. The sodium salts of the amino acids were prepared by mixing equivalent amounts of each one of them with 0.1 NNaOH solution and allowing the water to evaporate slowly in the water bath, washing the white residue with acetone, and drying it in a dry oven.
General Method for the Preparation of the Complexes cis- 1Pt(ino)z(am-ac)I Cl. 1 mmol of cis-Pt(ino)zCl;! and 1.2 mmol of the corresponding am-acNa were mixed in the solid state and 200 ml of methanol was added. The suspension was stirred at 30°C for 24 hr. It was then filtered through a filter paper from the unreacted material and the yellow filtrate evaporated to a small volume in the rotary evaporator. By adding an excess of acetone and ether (1: 1) to this solution a yellow material was obtained, which was filtered and dried in air. It was then recrystallized from a mixture of ethanol: water = 8:2 and dried first at room temperature and then at 110°C under vacuum. The yields varied from 50% to 75 % . General Method for the Preparation of the Complexes cis- 1Pt(ino)z(am-ac)Clj Cl.. 1 mm01 of each of the complexes cis-\Pt(ino)z(am-ac))Cl were disolved in the equivalent amount of an aqueous solution of HCl ( - 5 ml) and the mixture was left to react for about 16 hr at room temperature. After filtering the solution, the complexes were precipitated with isopropanol and diethylether (1: l), filtered, and dried first at room temperature and then at 110°C under vacuum. Yield was greater than 90%.
REFERENCES 1. C. Helene, Nucl. Acid Res. 2, 961 (1975). 2. H. Sigel, B. E. Fischer, and E. Farkas, Znorg. Chem. 22, 925 (1983).
78
A. Garoufis
et al.
3. M. Sabat, K. A. Satyshur, and M. Sundaralingam, J. Am. Chem. Sot. 105, 976 (1983). 4. A. Bere and C. Helene, Biopolymers 18, 2659 (1979). 5. 0. Yamauchi, J. Mol. Catal. 23, 255 (1984). 6. V. S. Krattiger, K. H. Scheller, and R. B. Martin, Znorg. Chim. Acta 59, 281 (1982). 7. S. H. Kim and R. B. Martin, J. Am. Chem. Sot. 106, 1707 (1984). 8. P. I. Vestues and R. B. Martin, Znorg. Chim. Acta 55, 99 (1981). 9. 0. Yamauchi and A. Odani, J. Am. Chem. Sot. 103, 391 (1981). 10. A. Odani and 0. Yamauchi, Znorg. Chim. Acta 66, 163 (1982). 11. A. Odani and 0. Yamauchi, BUN. Chem. Sot. Jpn. 54, 3773 (1981). 12. A. Odani, S. Deguchi, and 0. Yamauchi, Znorg. Chem. 25, 62 (1986). 13. J. P. Laussac, M. Pasdeloup, and N. Hadjiliadis, J. Znorg. Biochem., in press. 14. N. Hadjiliadis and T. Theophanides, Znorg. Chim. Acta 16, 77 (1976). 15. T. Giraldi and T. M. Taylor, Biochem. Pharmacol. 23, 1659 (1974). 16. H. C. Harder, R. G. Smith, and A. F. Leroy, Cancer Res. 36, 3821 (1976). 17. B. Rosenberg, Cis-Platin. Current Status and New Developments, A. W. Prestayko, S. T. Crooke, and S. K. Carter, Eds., Academic, New York, 1980, Chap. 2, p. 9. 18. J. P. Maquet, J. L. Butour, N. P. Johnson, H. Razaka, B. Salles, C. Vienssens, and M. Wright, Platinum Coordination Complexes in Cancer Chemotherapy (Proceed. 4th Int. Symp.) 1983, Vermont, USA, p. 27. 19. A. J. Repta and D. F. Long, Cis Platin. Current Status and New Developments, A. W. Prestayko, S. T. Crooke, and S. K. Carter, Eds., Academic, New York, 1980, Chap. 18, p. 283. 20. A. J. Charlson and W. A. Shorland, Znorg. Chim. Acta 93, L67 (1984). 21. L. Kumar, N. P. Kandasamy, T. S. Srivastava, A. J. Amonkar, M. K. Adwankar, and M. P. Chitnis, J. Znorg. Biochem. 23, 1 (1985). 22. L. Kumar, N. R. Kandasamy, and T. S. Srivastava, Znorg. Chim. Acta 67, 139
(1982). 23. S. Kasselouri,
A. Garoufis, and N. Hadjiliadis, Znorg. Chim. Actu, Bioinorg. Chem. Sec. 135, L3 (1987). 24. L. E. Erickson, J. W. McDonald, J. K. Howie, and R. P. Clow, J. Am. Chem. Sot.
90, 6371 (1968). 25. J. H. J. den Hartog, C. Altona, I. C. Chottard, I. P. Girault, J. Y. Lallemand, F. A. A. M. de Leeuw, A. T. M. Marcelis, and J. Reedijk, Nucl. Acid Res. 10, 4715 (1982). 26. M. Polissiou,
M. T. Phan Viet, M. St. Jaques, and T. Theophanides.
Znorg. Chim.
Acta 107, 203 (1985). 27. K. Okamoto, V. Behnam, M. T. Phan Viet, M. Polissiou, J. Y. Gauthier, S. HanesSian, and T. Theophanides, Znorg. Chim. Acta 123, L3 (1986). 28. K. Okamoto, V. Bahnam, J. Y. Gauthier, S. Hanessian, and T. Theophanides, Znorg Chim. Acta 123, L, (1986). 29. M. Polissiou, M. T. Phan Viet, M. St. Jaques, and T. Theophanides, Can. J. Chem. 59, 3297 (1981). 30. J. M. Neumann, S. Tran-Dinh, J. P. Girault, J. C. Chottard, T. Huynh-Dinh, and J. Igolen, Eur. J. Biochem. 141, 465 (1984). 31. J. H. J. Den Hartog, C. Altona, G. A. Van der Marel, and J. Reedijk, Eur. J. Biothem. 147, 371 (1985). 32. H. A. Tajmir-Riahi, M. J. Bertrand, and T. Theophanides. Can. J. Chem. 64, 960
(1986). 33. J. M. Neumann, S. Tran-Dinh, J. P. Girault, J. C. Chottard, T. Huynh-Dinh, Igolen, Eur. J. Biochem 141, 465 (1984). 34. C. H. Lee and R. H. Sarma, J. Am. Chem. Sot. 98, 3541 (1976). 35. C. H. Kim and R. H. Sarma, J. Am. Chem. Sot. 100, 1571 (1978).
and J.
INTERACTIONS
OF cis-pt(ino)$&
WITH AMINO
ACIDS
79
36. C. H. Lee, F. S. Ezra, N. S. Kondo, R. H. Sarma, and S. S. Danyluk, Biochemistry 15,3627 (1976). 37. F. S. Ezra, C. H. Lee, N. S. Kondo, S. S. Danyluk, and R. H. Sarma, Biochemistry 16, 1977 (1977). 38. A. R. I. Munns and P. Tollin, Acfa Crystallogr. B 26, 1101 (1970). 39. U. Thewalt, C. E. Bugg, and R. E. Marsch, Actu Crystullogr. B 26, 1089 (1970). 40. N. Nagashima and Y. Iitaka, Acta Cry.stalfogr. B 24, 1136 (1968). 41. T. J. Kistenmacher, C. C. Chiang, P. Chalilpeyil, and L. G. Marzilli, J. Am. Chem. Sot. 101, 1143 (1979). 42. C. C. Chiang, T. Sorrell, T. J. Kisternnacher, and L. G. Marzilli, J. Am. Chem. SOC. 100, 5102 (1978). 43. M. Tsuboi, K. Onishi, I. Nakagawa, T. Shimanouchi, and S. Mizushima, Spectrochim. Acta 12, 253 (1958). 44. K. Nakamoto, IR and Raman Spectra of Inorganic and Coordination Compounds, 3rd Ed., Wiley, New York, 1978. 45. J. F. Jackovitz, J. A. Dwekin, and J. L. Walter, Spectrochim. Acta 23A, 67 (1967). 46. I. Nakagawa, R. J. Hooper, J. L. Walter, and T. J. Lane, Spectrochim. Actu 21, 1 (1965). Received February 24, 1987; accepted April IS, 1987
MP, JPL.
ABSTRACT The reactions of ci.s-Pt(ino)2C12 with the amino acids Gly, L-Ala, L-Val, L-Ileu, L-Phe and L-Pro were studied in methanolic solutions. The (1: 1) adducts of the formulas cis-[Pt(ino)z(am-ac)]C1 were isolated from these reactions in the solid state, which in turn produce the cis-[Pt(ino)z(am-acH)C1]C1 complexes, by treating the former with equivalent amounts of HCl, in aqueous solutions. The complexes were characterized with elemental analysis, conductivity measurements, IR, ‘H NMR, and 13C NMR spectra. The results show that the purine ring of inosine interacts with the aliphatic side chain of the amino acids. The platination increases the percentage of the C3 f -endo-anti conformation of the sugar part of inosine.
INTRODUCTION Nucleic acids and proteins can recognize each other selectively in biological systems and interact through specific amino acid side-chains and nucleic acid bases [ 11. These specific interactions can in several cases be mediated through a metal ion, helping in the formation of ternary complexes [2, 31. For example, Cu+ z and Zn+’ ions can provoke interactions between polypeptides containing tyrosine and glutamic acid fragments and polynucleotides [4]. The various ligand-ligand interactions may be classified as (i) steric hindrance, (ii) electrostatic interactions and hydrogen bonds, (iii) hydrophobic interactions, and (iv) metal ion mediated electronic interactions [5]. Sigel et al. [l] studied the ternary complexes formed between ATP, the metal ions Mn+2, CU+~, Zn+2, Cd+2, and Pbf2 and various amino acids with increasing aliphatic
Address reprint requests to Professor Laboratory of General and Inorganic
N. Hadjiliadis, University of Ioannina, Chemistry, Ioannina 45-332, Greece.
Department
Journal of Inorganic Biochemistry 31, 65-79 (1987) @ 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
of Chemistry,
65 0162-0134/87/$3.50
66 A. Garoufis et al.
side-chains. They found that hydrophobic interactions existed between the aliphatic side chains of the amino acids and the aromatic purine ring of ATP, which increased with the length of the chain. Similar studies between various peptides and nucleotides have also been carried out by Martin et al. [6-81, who observed similar ligand-ligand interactions. Yamauchi et al. [5, 9-121 also found similar ligand-ligand interactions in mixed amino acid-metal complexes. The same is true for the recently published PdGHL-5 ‘-IMP and 5 ‘-GMP ternary system [13], where, based on the ‘H NMR spectra, a very strong interaction between the imidazole ring of the peptide and the purine ring of the bases, was observed. In the present paper we describe the interaction of cis-Pt(ino)2Clz, a compound of cis geometry with possible antitumor activity [14], and various amino acids in methanolic solutions. This is part of a more general project of ternary complexes of Pt +2 or Pd+2, nucleosides or nucleotides, and amino acids or peptides undertaken by us in order to detect and characterize such ligand-ligand interactions. In addition, such studies are also interesting because of the anticancer properties of cis-DDP. Despite the fact that cis-DDP interacts with DNA during its replication, thereby demonstrating its anticancer properties [ 15-171, its interaction with the other chemical constituents of the body is not excluded and has not yet been elucidated [ 181. For example, cis-DDP reacts with certain small molecules of the blood plasma, producing complexes of low molecular weight, essential for the availability and the elimination of Pt(I1) from the organism [ 191, according to the reaction Protein-Pt
* complex-t
Protein + Pt-small
molecules
(1)
Finally, the new mixed complexes of Pt + 2 with amino acids and nucleosides having the cis configuration might also be potential anticancer agents, as in other similar cases [20-221. Several of the complexes are currently under screening against various tumors.
RESULTS
AND DISCUSSION
The amino acids glycine, L-alanine, L-valine, L-isoleucine, t_-proline, and Lphenylalanine, in their sodium salt forms, were used in this study to prepare 1: 1, N,O chelate complexes with cis-Pt(ino)2C12 [23], in methanolic solutions, according to cis-Pt(ino)2C12 + am - ac - Na CHZoHcis-[Pt(ino)s(am-ac)]Cl + 0 II The Pt-O-Cbond of the amino acids, in these complexes, in 0.1 N HCl solution, without breaking of the Pt-NH2-bond: cis-[Pt(ino)2(am-ac)]C1
NaCl
(2)
could be easily broken
“z’ cis-[Pt(ino)2(am-acH)C1]C1
(2)
The elemental analyses of the 12 complexes obtained in this way agree with their empirical formulas and are given in Table 1. The complexes of both series are 1: 1 electrolytes as expected, in aqueous solutions, and the values of the molar conductivities are also included in Table 1. The structures and the conformation of the complexes in the above series are mainly deduced from the NMR and IR spectra, which are briefly discussed below.
INTERACTIONS OF cis-Pt(ino)rC& WITH AMINO ACIDS
TABLE 1. Elemental
Analysis and Conductivity
Complexes
cis-[Pt(ino)s(Gly)]Cl
cis-[Pt(ino),(Ileu)]Cl
cis-[Pt(ino),(Phe)]Cl
ck-[Pt(ino),(GlyH)Cl]Cl
cis-[Pt(ino),(AlaH)Cl]Cl
cti-[Pt(ino),(ValH)Cl]Cl
cis-[Pt(ino)#roH)ClJCl
ci.s-[Pt(ino),(Ileu)Cl]Cl
Pt(%)
67
Values of the Complexes
Cl(%)
C(W)
H(%)
N(%)
Calc
23.19
4.21
31.41
3.33
14.98
Found
23.03
3.87
31.40
3.50
14.70
Calc
22.81
4.14
32.28
3.51
14.73
Found
22.40
3.80
31.92
3.22
14.65
WC.
22.09
4.01
33.97
3.85
14.27
Found
21.85
3.65
33.61
3.98
14.20
Calc.
22.14
4.02
34.05
3.63
14.30
Found
21.15
4.20
33.90
3.85
14.21
Calc.
21.74
3.95
34.78
4.01
14.04
Found
21.41
4.32
34.65
4.25
13.85
talc.
20.95
3.80
37.31
3.65
13.53
Found
20.58
4.16
36.98
3.53
13.75
Calc.
22.25
8.09
30.12
3.31
14.37
Found
23.00
8.16
29.81
3.45
14.15
talc.
21.90
7.96
30.99
3.48
14.15
Found
21.83
8.51
30.85
3.58
14.27
talc.
21.23
7.72
32.66
3.81
13.72
Found
21.91
7.95
32.35
4.05
13.62
Calc.
21.28
7.73
32.73
3.60
13.75
Found
21.41
7.91
32.55
3.48
13.59
Calc.
20.92
7.60
33.45
3.91
13.51
Found
21.25
7.42
33.15
4.15
13.32
mole-‘)
116
113
101
105
103
123
90
98
98
93
97
rH NMR and 13C NMR Spectra The spectra were recorded in DrO solutions and the chemical shifts are given in Table 2. The N7 coordination of inosine in cis-Pt(ino)$& [ 141 is retained in the complexes of both series, since the Hs proton is always shifted downfield more than the Hz proton and 0.7-0.9 ppm, as compared to the free ligand. The chemical shifts of the amino acid protons in the complexes are very small compared to the free ones. Sigel et al. [2] observed upfield shifts of the amino acid protons, with aliphatic side-chains, in ternary complexes with ATP, and explained the phenomenon as due to ligand-ligand hydrophobic interactions. These interactions were stronger as the size of the aliphatic side-chains of the amino acids was increased. In our ternary systems, the chemical shifts of the amino acid protons are the average of downfield shifts due to Pt(II) coordination and upfield shifts due to hydrophobic
8.055
8.145
8.152
8.165
8.830
8.891
8.902
8.912
cis-[Pt(ino),(Gly)Cl]Cl
cis-[Pt(ino),(Val)Cl]Cl
cis-[Pt(ino),(Ileu)Cl]CI
6.106
6.095
6.087
6.048
6.043 6.049
6.003 6.086 6.060
4.624
4.616
4.604
4.622
4.611 4.619
4.688 4.604 4.603
4.417
4.408
4.399
4.400
4.391 4.400
4.376 4.396 4.392
4.248
4.249
4.252
4.238
4.227 4.239
4.217 4.249 4.230
3.962
3.954
3.945
3.927
3.920 3.931
3.869 3.942 3.930
3.852
3.845
3.836
3.815
3.808 3.814
3.779 3.833 3.821
1.039 AS = + 0.014 0.986 1.014 AS = + 0.020 0.938 = +0.028
0.975 A6 = + 0.003 1.025 0.990 A.6= i-0004 0.910 A&= -0.008
1.479 A6 = + 0.029
3.574 A6 = + 0.047
1.456 AIS= +0.006
3.539 A6= +0.012
CHz
3.610 A6 = + 0.024 3.679 Ah= +0.043
2.010 A.6= +0.060
3.651 A&= +0.015
3.59 A6 = + 0.004
CH,
2.275 Ad = + 0.030
1.955 A&= +0.005
2.25 AS = + 0.005
CHo
Amino acid protons
Academic, New York, 1971. The positive shift signifies down field shift and the negative one uptield shift relative to the free amino acids.
in chemical shift from the positions of the free amino acids, taken from A. F. Casy, PMR Spectroscopy in Medicinal and Biological Chemislry,
8.055 8.055
8.833 8.830
cis-[Pt(ino),(Pro)]Cl cis-[Pt(ino)2(Val)]C1
u AS. The difference
8.123 8.144 8.089
8.251 8.888 8.855
Inosine cis-Pt(ino)zC12 cis-[Pt(ino),(Gly)]Cl
Complexes
in ppma ~._____
Inosine protons ___.__
TABLE 2. ‘H NMR Chemical Shifts of the Complexes
INTERACTIONS
OF cis-Pt(ino)rQ
WITH AMINO
ACIDS
69
interaction of the side-chain of the amino acids with the aromatic purine rings of the two inosine molecules. The result is a very small downfield shift (0.003-0.015 ppm) in most cases in the chelated complexes cis-[Pt(ino)(am-ac)]Cl. An upfield shift of 0.008 ppm is observed only in the case of the complex with Ileu, in one of its -CH3 resonances. The existence of a synergic effect of upfield and downfield shifts is obvious in the present system, since the downfield shifts due to coordination to Pt(I1) are much larger in binary Pt(II)-amino acid complexes, ranging from 0.2 to 0.8 ppm [24]. This implies a hydrophobic ligand-ligand interaction. This interaction is even larger in the chelated N,O than in the nonchelated series, since the downfield chemical shifts are larger in the latter. This might be the result of a more fixed position in space of the aliphatic side-chains in the former series, bringing it closer to the purine rings and hence to a stronger interaction. In the nonchelated species, on the other hand, this group can be a little further from the purines as subject to free rotation of the amino acid around the Pt-NH2 bond. The ligand-ligand hydrophobic interaction is also shown from the upfield shift by 0.05 ppm of the Hs and 0.089 ppm of the H2 proton of inosine, in the ternary N,O chelated systems, with L-proline, L-valine, and t_-isoleucine. In the second series of complexes, on the other hand, very slight downfield shifts of 0.003-0.024 ppm for the Ha protons and of 0.001-0.021 ppm for the H2 protons are observed. Upfield shifts are also observed for the Hi g protons of the sugar part of inosine, in the first series of complexes by 0.026-0.042 ppm and in the second downfield shifts by 0.001-0.020 ppm. Strong ligand-ligand interaction was also observed in the GHL-Pd(II)nucleotide system [13], though between aromatic rings. The t3C NMR spectra of the complexes cis-[Pt(ino)2(Ileu)]C1 and cis-[Pt(ino)z(IleuH)C1]C1 were recorded in D20 solutions and confirm the N7 coordination of inosine in both compounds, the N,O chelation of Ileu in the first, and the N coordination of Ileu in the second. Thus, the Ca near the coordination site N7 of inosine shifts downfield in the first complex by 4.16 ppm and by 5.56 ppm in the second, while the C5 atom shifts upfield by 1.42 ppm in the first and by 1.00 ppm in the second. The shifts of the other carbons are given in Table 3. Although the carbonyl carbon resonance is not seen in the I3C NMR spectra of both complexes, the C, of Ileu (near the -COOgroup) shifts by 1.60 ppm in the complex cis-[Pt(ino)z(Ileu)Cl]Cl and by 1.92 ppm in the cis-[Pt(ino)2(Ileu)]Cl, 0 II indicating Pt-O-Cbonding in the latter complex. The influence of Pt(II)-inosine and amino acid coordination to the conformation of the sugar moiety of the nucleoside was further explored, by measuring the coupling constants of the various sugar protons in the ‘H NMR spectra [25-331. The furanose ring of the sugar in many nucleosides is in equilibrium between the Cj’-endo, anti = 3E and the C2 I -endo, anti = z E conformations (Figure la) and metal coordination to the bases shifts the equilibrium towards the former [25-331. The conformation around the C(4’)-C(5’) bond, on the other hand, described as gg, gt or tg [34-371, was found not to favor the gg conformer on passing from G5 ‘p to its complex with K,PtCI, [29], while it is favored in its complex with cis- and transPt(NH3)$12 [26-281 (Figure lb). A method for the calculation of the various sugar conformers of nucleosides, based on the ‘H NMR spectra, was first proposed by Sarma et al. [34-371 and is based on the estimation of the coupling constants 3J nn and application of the Karplus equation. The
-
157.66
-
160.40
c6
-
149.19
-
151.88
c2
-~-
-
146.93
150.06
150.06
c4
-
139.89
145.45
144.05
c8
CS
-
125.32
124.32
123.90
__~_______~
Purine
Inosine
-
88.73
92.00
91.87
C,f
-
86.62
87.91
88.03
C4’
with Ileu, in ppm
-
75.13
77.18
76.98
CZ’
Sugar
-
11.22
72.30
72.49
Cjf
-
62.24
63.39
63.58
cg’
._ -_--.-_-
60.30
-
61.90
62.22
c,
36.50
-
38.43
38.50
c,
-.-.-.----.--_-
a Taken from A. J. Jones, D. M. Grant, M. W. Winkley, and R. K. Robins, J. Am. Chem. Sot. 92, 4079 (1970). b Taken from A. F. Casy, PMR Spectroscopy in Medicinal nnd Biological Chemistry, Academic, New York, 1971.
Isoleucineh
Inosine”
cti-[Pt(ino)2(Ileu)C1]C1
cis-[n(ino),(Iieu)]Cl
Compounds
_-
TABLE 3. 13C NMR Chemical Shifts of the Complexes
25.10
-
27.05
27.05
C”
15.30
-
17.16
17.29
c,
Amino acid G
11.70
-
13.65
13.65
--
174.60
-
-
co
INTERACTIONS
OF cis-Pt(ino)&
573
C&end0
WITH AMINO ACIDS
71
C&end0
a
“4, QQ
HO
<:a,
HO
“5‘
H4’ Ql
b FIGURE_ 1. The C3 f-end0 and the Cz I -endo sugar conformations, with 0 carbon and 0 oxygen atoms. (b) The sugar conformation around the C4t-C5 1 bond.
calculation of the gg, gt, or tg conformers around the C(4 ‘ )-C(5 ’ ) bond has also been given by Sarma [34]. The results for inosine, cis-Pt(ino)Q, and the various complexes of the type cis-[Pt(ino)2(am-ac)]Cl and cis-[Pt(ino)z(am-acH)Cl]Cl are summarized in Table 4, and typical spectra are shown in Figure 2. It is seen that the complexation of K$tC& to inosine favors the C3j endo anti conformation in the cis-Pt(ino)&& complex, increasing it to 59 % from 46% in the free ligand. The gg percentage is also favored in the complexes ranging from 71% to 80%, but not differing appreciably from the free ligand (74%). The amino acid complexations in the N,O chelated forms, do not affect the Kq = %3E/%2E significantly (Table 4). A slight decrease in the %3E is, however, observed in the glycine and proline complexes, while in the valine and isoleucine derivatives there is a further decrease of the %3 E endo, being 50% in both. This reflects the greater size of the amino acid aliphatic side-chains, producing larger interactions with the purine rings. It increases slightly again to 55 % in the nonchelated species. For the percent of the gg conformers the opposite effect is observed, e.g., slight decrease in the nonchelated complexes (See Table 4). It is worthwhile mentioning that the sugar pucker of inosine in the solid state is in the C3’ endo anti gt conformation [38]. Thewalt et al [39] found two independent inosine molecules in its crystal structure, the one assuming the CZ~ endo anti gg conformation and the other the C3/-endo anti gg conformation). The S/-IMP disodium salt on the other hand, has the C(2 ‘) endo anti gg conformation in the solid state [40]. The conformation of the sugar in the disodium salt of 5 ‘-IMP does not change in the crystal structure of a nonstoichiometric compound of cis-Pt(NHW12, being again the C(2 ‘) endo anti gg [41]. In a Cu(II) complex of 5 ‘-IMP disodium salt and dien, the same pucker of the sugar was once more found [42], while’ in a similar one with 2,2’-bipyridyl the C3 I endo anti gg conformation predominates [43]. Finally the t and g + h conformations around the R
I -CH,-CH,-
INTERACTIONS
5,O
8.0 FIGURE
OF cis-Pt(ino)*Q
2.
4,o
WITH AMINO
P P r1
ACIDS
73
281)
The 250 MHz, ‘H NMR spectrum of the complex cis-[Pt(ino)z(val)]C1.
bond of L-Val and L-Ileu were calculated, in the complexes with these two amino acids, from the values of the coupling constants JHaHBgiven in Table 5 and using JT = 13.3 Hz and JG = 2.4 Hz [6-81. The calculation of the individual h and g conformers is not possible in this case, since the amino acids do not possess a CH,,,-CH Z(ojsystem. The results are included in Table 5 and show an about 85 %-I36 % abundance of the h + g conformers for the Ileu complexes and 82%-88 % for the L-Val complexes. In similar Pd(I1) complexes with dipeptides containing L-Val or L-Ileu, the h + g conformers were predominant (90%) [8]. The same was true in similar mixed Pd(II)-depeptide nucleotide complexes [6]. In the GHL-Pd-nucleotide complex, on the other hand, the h conformer was more than 80 % [ 131. The increase of the percentage of h + g conformers in mixed Pd(II)-amino acid complexes has been interpreted as due to increase of the ligand-ligand hydrophobic interactions in these systems [9, 121. Thus the higher values of this sum in the complexes with L-Ileu than with L-Val may show a stronger such interaction in the first ones, as expected. IR Spectra In the region below 1800 cm-‘, several bands can be identified and help to better understand the structures of the complexes. The characteristic IR frequencies are given in Table 6. In the present study, many bands mainly due to the various -NH*, and -CH’motions of the amino acids were identified as follows: Glycine in its zwitterionic form, shows the v,(COO-) at 1610 cm-’ and the &(NH3+) at 1585 cm-’ [44,45]. In the complex TABLE 5.
‘H NMR Coupling Constants and Conformations around the -CH -CHR( bonds (x)(a) (B) 0)
g+h Complexes ci.+[Pt(ino)~(Val)]Cl cis-[Pt(ino),(ValH)Cl]Cl cis-[pt(ino)z(Ileu)]Cl cis-[F’t(ino)z(IleuH)C1]C1
JBX
4.359 4.325 3.975 3.902
(%o)
18 17 15 14
82 83 85 86
cis-lFY(ino),(Val)lCi
Ino
Complexes
c,
1600s
1396s
-
_
-
_
_
1625s
1330~1 1620s
_
163&h
541m
-
540m
113h
1118s
1128m
804m
804m
804m
1298m
1308m
_
_
_
1342s
160&h
1459m
-
1595s 156Om 1515s
1700s
1310s
1335m
1362s
1432m
VI’-Cl
1595s 156Om 1520~1
1600s
1705s
_
Gff-NCCO
_
16Ctsh
17Oas -
6(NHz) p,(NHz) pw(NHz) pw(CQO-)
1407s
6(CH)
1595s 156Om 152OW
&CHz) p&H,)
334w
S(CH3)
1591s 1555s 1519m
v,(CHJ
_.
1698s
v@mc-) amino acid
1592 155lm 1518111
C=N) ring
v(c =
1690s
acid
amino
v.(coo-)
IR Bands of the Complexes
u.(C=O) skeletal
TABLE 6. Characteristic
831m
832111
830m
828m
822m
INTERACTIONS
E
Fi OD 3
5 I
I
I I
x z I I
I
I
I
I
OF cis-Pt(ino)zClz
WITH AMINO ACIDS
75
76 A. Garoufis et al.
cis-[Pt(ino)(Gly)]Cl, the broad band at 1630 cm-’ is assigned to the overlapping of the coordinated to Pt(II), 6(NH2), and v,(COO-) of the amino acid [23]. The intensity of this band diminishes upon deuteration and a new weak band appears at - 1600 cm- ‘, obviously due to the metal coordinated v,(COO-) mode. On passing to the cis[Pt(ino)z(GlyH)C1]C1 complex, a band of medium intensity appears at 1634 cm-‘, which disappears upon deuteration, due to the metal coordinated &NH>) motion. On the other hand, the intensity of the band at 1700 cm- ’ due to the VC = 0 of the keto group at the sixth position of inosine, is enhanced, since it coincides now with the v,(COOH) group [23]. The same behavior is also observed in all the other complexes, confirming the N,O chelation of the amino acids in the first series and their N coordination in the second. In a few cases the &NH*) and the v,(COO-) of the metal coordinated amino acids are shown as two different bands, Such is the case with L-ala, in the complex cis[Pt(ino)2(ala)]C1 showing the 6(NH2) at 1629 cm-. ’ and the v,(COO-) at 1597 cm- ‘. The metal coordinated v,(COO-) of the complex cis-[Pt(ino),(Gly)]CI is assigned to a new band at 1407 cm-~ I, while it occurs at 1412 cm- I in free Gly [44]. It is shown at 1362 cm- 1in the complex with L-ala, possibly coupled with other motions. It occurs at 1374 cm-’ in the Pt(L-ala)z complex [45]. It is also seen at 1342 cm--’ in the corresponding complex with t_-val [46]. at 1338 cm-’ in the complex with L-pro, and 1383 cm-’ in the complex with L-Ileu. All these bands disappear in the second series of complexes indicating again the existence of free -COOH groups in them. The A(Y, - YJ of the -COOgroup varies from 223 to 267 cm ’ in the various Pt(I1) complexes, in agreement with other similar complexes [45, 461. The 6(-CH) and p(-CH) of the various aliphatic groups of the amino acids can be identified in the region of 1250- 1450 cm - I. These bands shift only slightly in the two series of complexes, compared to the free amino acids, since they are rather far from the coordination sites. More such bands are identified in the complexes with L-Val and L-Ileu, containing more aliphatic side-chains (See Table 6). A band near 1300 cm -~’ appearing constantly in all the complexes of the first series and disappearing in the second, should be associated with a deformation motion of the chelated five-membered , N-C
I ’ o-c
Pt
ring. The same is true with the band at 804 cm- I, which disappears in the second series of complexes and which may be assigned to the p,(COO- ) [45, 461. A band near 540 cm- ’ that appears in the complexes of both series disappears upon deuteration and is assigned to the metal coordinated &NH,) motion. A medium intensity band near 330 cm- ’ appears in the second series of complexes cis-[Pt(ino),(am-acH)Cl]Cl and is assigned to the vPt-Cl, since it is absent in the first series of complexes, with N,O chelated amino acids cis-[Pt(ino)2(am-ac)]Cl. Finally, inosine shows a band at 822 cm-‘, which shifts to 828 cm ’ in the complex cis-Pt(ino)$l;! and the second series of complexes and at 830 cm ’ in the first series. This has been assigned to a sugar-ring vibrational mode and might be related to the increase of the percentage of the C 3j-e&o-anti conformer of inosine, on passing to its Pt(I1) complexes [28. 29, 331. In the case, however, of guanosine. the opposite effect was observed, e.g., removal of the 824 cm- I of the free ligand, with a higher
INTERACTIONS
OF cis-Pt(ino)Q
WITH AMINO
ACIDS
77
percentage of the CZ I-endo-anti conformer, to 797-798 cm-’ in its Pt(I1) complexes with increased proportion of the C3 ‘-endo-anti conformer [28, 29, 331. EXPERIMENTAL Materials Glycine, L-Alanine, L-Valine, L-Isoleucine, L-Proline, L-Phenylalanine, and Inosine were purchased from Aldrich Chemical Company and Fluka A.G. Potassium tetrachloroplatinate was a gift of Degussa AG (West Germany). Methods (i) The elemental analyses of Pt and Cl were performed in the laboratory of Inorganic Chemistry at Ioannina, while those of C, H, and N were performed in the Laboratoire de Chimie de Coordination, in Toulouse. (ii) The conductivity measurements were performed in an E365B Conductoscope, Metrohm Ltd., Herisau, Switzerland. (iii) The IR spectra were recorded on a Perkin Elmer model 580 spectrophotometer. (iv) The ‘H NMR spectra were obtained on a Brucker WM-250 spectrometer equipped with an Aspect 3000 computer. (v) 13C NMR spectra were recorded at 22.63 MHz on a Brucker WH-90-NMR spectrometer, operating in the pulse Fourier transform mode. Both ’ H and 13C chemical shifts were measured in parts per million with DSS as reference. Preparation
of the Compounds
The starting material cis-Pt(ino)&lz was prepared from K,PtCI, and inosine, according to a published method [ 141. The sodium salts of the amino acids were prepared by mixing equivalent amounts of each one of them with 0.1 NNaOH solution and allowing the water to evaporate slowly in the water bath, washing the white residue with acetone, and drying it in a dry oven.
General Method for the Preparation of the Complexes cis- 1Pt(ino)z(am-ac)I Cl. 1 mmol of cis-Pt(ino)zCl;! and 1.2 mmol of the corresponding am-acNa were mixed in the solid state and 200 ml of methanol was added. The suspension was stirred at 30°C for 24 hr. It was then filtered through a filter paper from the unreacted material and the yellow filtrate evaporated to a small volume in the rotary evaporator. By adding an excess of acetone and ether (1: 1) to this solution a yellow material was obtained, which was filtered and dried in air. It was then recrystallized from a mixture of ethanol: water = 8:2 and dried first at room temperature and then at 110°C under vacuum. The yields varied from 50% to 75 % . General Method for the Preparation of the Complexes cis- 1Pt(ino)z(am-ac)Clj Cl.. 1 mm01 of each of the complexes cis-\Pt(ino)z(am-ac))Cl were disolved in the equivalent amount of an aqueous solution of HCl ( - 5 ml) and the mixture was left to react for about 16 hr at room temperature. After filtering the solution, the complexes were precipitated with isopropanol and diethylether (1: l), filtered, and dried first at room temperature and then at 110°C under vacuum. Yield was greater than 90%.
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INTERACTIONS
OF cis-pt(ino)$&
WITH AMINO
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79
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