Ternary Complexes of cis-(NH3)2PtCl2 (cis-DDP) With Guanosine (guo), Cytidine (cyd) and the Aminoacids Glycine (gly), L-Alanine (ala), L-2-Aminobutyric Acid (2-aba), L-Norvaline (nval) and L-Norleucine (nleu)

The ternary complexes of formulae cis-[(NH3)2Pt(nucl)(amac)]NO3, where nucl = guo and cyd (guanosine and cytidine) and amac = the deprotonated aminoacids glycine (gly), L-alanine (ala), L-2-aminobutyric acid (2-aba), L-norvaline (nval) and L-norleucine (nleu), were prepared from the reactions of the binary chelated ones cis-[(NH3)2Pt(nucl)(amac)]NO3 with the nucleosides. They were characterized by 1H, 13C and 195Pt NMR and IR spectra, together with elemental analysis and conductivity measurements. The aminoacids coordinate with Pt(II) in the ternary complexes with their terminal -NH2 groups, guo through N7 and cyd through N3. Ligand-ligand hydrophobic interactions were also observed in the ternary complexes and were stronger with longer aliphatic chains of the aminoacids. The 3E sugar conformation increased by 5-7% in the ternary systems, as compared to the free nucleosides, while the percentage of the gg conformation remained almost constant and the one of the anti conformation of the sugar increased also slightly. Finally, the h conformer around the Cα-Cβ bonds of the aminoacids reached a maximum in the binary systems and decreased again considerably in the ternary ones.

Sugar conformations and ligand-ligand interactions of the bases and aminoacids-peptides, simultaneously coordinated with Pt(ll) were examined in these studies. For example, the hydrophobic ligand-ligand interactions are usually stronger with the increase of the aliphatic side chain of the aminoacids-peptides, but are usually weaker with increasing distance from the bonding sites. They are stronger in the cis-rather than the trans-series of complexes [3,7,8,10]. Also, the anti conformation of the sugar was increasing in the ternary complexes, compared to the free ligands and it was larger in the transthan the cis-ternary complexes.
The present paper concludes our studies on such simple ternary systems. It reports on complexes of the general formulae cis-[(NH)Pt(nucl)(amac)]NO, where nucl is guo or cyd (Structure I) and amac the conjugated bases of the aminoacids glycine, L-alanine, L-2-aminobutyric data points were used. A line broadening factor of 0.3 Hz was used in processing the data. aC NMR spectra were recorded on the same spectrometer (Brucker AMX-400 MHz) at 100.62 MHz. A sweep width of 22727 Hz, 2500-3000 scans (sample concentration 20 mM) and 16K data points were used, as well as a line broadening factor of 2.5 Hz in processing the data.
Pt NMR spectra were recorded on the Brucker AMX-400 MHz spectrometer at 86.02 MHz. Shifts are reported relative to KPtCI (external standard 1630 ppm for KPtCI). Spectra were run typically with 40000 transients (sample concentration 20 mM) and a spectral width of 125 KHz. A pulse duration of 35.6 ms, followed by aquisition time of 65 ms and a delay time of 0.1 s was used. A line broadening factor of 25 Hz was used in the processing the data.
1.5 mmoles of guo or cyd were dissolved in about 100 ml of water under stirring at 60C. mmole of the complex cis-[(NH,)Pt(amac)]NO was added to the solution and this was stirred for 48 h at 60C, in the dark. The pH of the solution was kept at 5 during the reaction, by addition of a 10 M solution of HNO. After cooling to 0C and filtration of the unreacted guo, the solution was concentrated to ml and passed through a Sephadex column (G-10, Pharmacia, 60 x 1.5 cm) using water as eluent. Fractions of 2 ml were collected. The ternary complexes of guo were eluted after unreacted binary ones (cis-[(NH)Pt(amac)]NO). Ocassionally, a second elution was required, in order to get pure complexes in larger amounts. The yields were varying 15-40 %. Deuterated Complexes These were prepared by dissolving the complexes in DO, followed by lyophilization.

Results and Discussion
The complexes were prepared according to the general reactions:

Erasmia Katsarou, Costas Charalambopoulos and Nick Hadjiliadis
Metal-Based Drugs The elemental analyses are in agreement with the general formulae of the complexes. The molar conductance values confirm the 1:1 electrolytic nature of the complexes.
Binding Sites

H-NMR
The chemical shifts of all compounds are included in Table I. The He proton of guo in the ternary complexes shifts downfield by about 0.6 ppm compared to the free ligand, while the H and H aromatic protons of cyd by only 0.07-0.10 ppm, indicating N and N coordination with the metal, of guo and cyd respectively [11,[16][17][18][19]. These shifts are smaller than the ones caused by cis-and trans-DDP bound to the same nucleosides of about ppm [17,19]. It was explained by the hydrophobic ligand-ligand interactions, taking place between the aminoacids and the nucleosides, bound simultaneously to the same metal [7,8,11].

C-NMR
The results obtained from the H-NMR spectra on the binding sites are confirmed by the C-NMR spectra of the compounds [20,21]. All C-NMR chemical shifts of the free aminoacids, the binary and the ternary complexes are given in Table I1.
In the ternary complexes with guo all nucleoside carbon atoms are shifted downfield compared to the free ligand, with the most downfield shifted the Ce atom (..7 ppm), near the N binding site with Pt(ll) of guo [20][21][22]. (See Table 2). In the cyd ternary complexes on the other hand, the C atom near the binding site is shifted upfield by ..3 ppm, thus confirming the N coordination with Pt(ll) in solution [23,24]. The other base carbon atoms are not considerably shifted on passing from free cyd to its ternary complexes. It is worthwhile mentioning that most of cyd carbon atoms including the sugar are observed as doublets in the ternary complexes (Table II), thus confirming the hindered rotation around the Pt-N bond, observed also in the H-NMR spectra [24].
The aminoacid carbon atoms on the other hand, are also not considerably shifted in the complexes, except the C atoms near the -NH coordination site with Pt(ll) (2-3 ppm on passing from the free aminoacids to the binary complexes and ..6 ppm to the ternary ones) [11]. Finally, the terminal carboxylate carbons of the aminoacids shift by about 15 ppm on passing from the free aminoacids to their chelates with Pt(ll), but only by about 3 ppm in the ternary complexes, thus confirming the non existence of the Pt-O bonds in the latter case.
The Further in the guo ternary systems a decrease in the intensity of the band at 825 cm is observed, together with an increase of the one at 802 cm , compared to the free guo. This indicates an increase of the percentage of the C.-endo (E) sugar conformation in the complexes, as it is also suggested from the H-NMR spectra (See Sugar Conformation). VoL4,No.2,1997 Ternary Complexes ofcis-(NH3) 2 PtCl 2 (cis-DDP) with Guanosine (guo), Cytidine (cyd) and the Amino Acidv Glycine (gly), L-Alanine (ala), L-2-Aminobutyric Acid (2-aba), L-Norvaline Oval) and L-Norleucine (nleu) Ligand-L. igand Interactions 'H-NMR spectra reveal the existence of hydrophobic ligand-ligand interactions in solution. Thus, the chemical shifts of the o-aliphatic protons of the aminoacids near the bonding sites with Pt(ll), are observed upfield by about 0.7 ppm, on passing from the binary to the ternary complexes [8,18,19,28]. The shifts of the other aminoacid protons are also upfield in the ternary complexes, compared to the binary ones, but decreased with distance from the bonding sites (See Table I).
More particularly, the difference in chemical shifts, A5=5 amacH(free)-5 amac(tern.complex) between the zwitterionic forms of the aminoacids and their ternary complexes, given in Table IV, shows quantitatively the strength of ligand-ligand interactions [3,8,9]. They are larger in the case of guo complexes than cyd (See Figure 1). Table V contains the difference in the chemical shifts of the terminal methyl groups of the anionic forms of the aminoacids and their ternary Pt(ll) complexes, AS(ppm)=Samac-Stern.complex. The more positive A5 values, the stronger the hydrophobic ligand-ligand interactions [8,28,29]. Thus,they increase with longer aliphatic side chain and they are stronger in the ternary guo complexes than in the cyd ones, as expected. Like in other similar cases [8,11], a hindered rotation around the Pt-N bonding of the ternary complexes with cyd is also observed in the present system, except the complex containing also gly (See Figure II). Both H and Hs of cyd are shown up as two doublets in all the other cases containing chiral aminoacids [30-32] and it persists even at 90C. Two sets of signals are also observed for the Hi. sugar protons of cyd and two triplets for the & protons of the aminoacids (except glycine). This hindered rotation around the Pt-N bonding was assigned to two diastereoisomers of head to tail oriented nucleobases and due to ortho substituents of cytidine [30,33].

Sugar Conformation
Estimation of the J,, coupling constants from the H-NMR spectra and application of the  The percentage of the gg conformation around the C.-C. bond on the other hand, remains almost constant in the ternary complexes, compared to free guo, as it is also true when the ligand was coordinated to cis-DDP [38,39]. However, it was found to decrease slightly in other similar systems [2,7,11]. In the case of the ternary complexes with cyd on the other hand, it decreases by 4-9% (See Table 6).
The percentage of the syn-anti conformations can be calculated from the relationship 5os P,,6, + Pant)anti (7), with & the chemical shift of the Hz proton, assuming 100% syn conformation for Vol. 4,No.2,1997 Ternary Complexes ofcis-(NH3) 2 PtCl 2 (cis-DDP) with Guanosine (guo), Cytidine (cyd) and the Amino Acids Glycine (gly), L-Alanine (ala), L-2-Aminobutyric Acid (2-aba), L-Norvaline (nval) and L-Norleucine (nleu) Conformation Around the C-C Bond of the Aminoacids The percentage of the three possible conformations around the C-C bond, g, h, t, designed in Figure 4 for aminoacids chelates, could be calculated in the case of the ternary complexes with cyd, where the sum of the coupling constants J+Jac (Hz) [8,9,11,40] could also be calculated. In the case of the ternary complexes with guo, this sum could not be calculated, due to overlapping with sugar protons. The results are included in Table VII.  [8,9,11], the percentage of the h conformer which directs the aliphatic side chain towards the metal ion (Fig. IV), increases first on passing from the free aminoacids to their chelates with Pt(ll), by about 10-20%. However, on passing to the ternary complexes, a decrease of about 19-24% of the h conformer is again observed, with values lower than the ones of the free ligands. This is possibly due to the monocoordination of the aminoacids with Pt(ll) through the terminal-NH group alone and to the ligand-ligand interactions taking place in the ternary complexes.