Comparison of N-acetylmethionine reactivity between oxaliplatin and an oxaliplatin derivative with chiral (S,S) amine nitrogen atoms

Article abstract of DOI:10.1016/j.ica.2013.03.008

We have synthesized an oxaliplatin derivative using N,N′-dimethyl-1, 2-diaminocyclohexane (Me₂dach) as the diamine ligand. The complex (S,R,R,S)-Pt(Me₂dach)(oxalate), where S,R,R,S represents the chiralities at N,C,C,N, respectively, was prepared and characterized by ¹H NMR spectroscopy, COSY, NOESY, and HMQC. Oxaliplatin reacts with N-acetylmethionine (N-AcMet) to form [Pt(dach)(N-AcMet-S)₂] and [Pt(dach)(N-AcMet-S,N)], with the former favored at higher molar ratios of N-AcMet. In contrast, Pt(Me₂dach)(oxalate) reacts to form [Pt(Me ₂dach)(N-AcMet-S,O)]⁺ even in the presence of excess N-AcMet. Molecular mechanics calculations are consistent with significant steric clashes in models of [Pt(Me₂dach)(N-AcMet-S)₂]. When N-AcMet was reacted with an excess of each platinum complex, the rate of N-AcMet decrease was very similar for both complexes. Thus, the methyl groups at the nitrogen atoms had little to no effect on the addition of the sulfur atom of a single N-acetylmethionine, but they prevented chelation of the amide nitrogen or coordination of a second N-acetylmethionine residue.

Full text of DOI:10.1016/j.ica.2013.03.008

Accepted Manuscript
Comparison of N-acetylmethionine reactivity between oxaliplatin and an oxa‐
liplatin derivative with chiral (S,S) amine nitrogen atoms
Kevin M. Williams, Amy D. Poynter, Jonathan D. Hendrie, Daniel C. Jackson,
Virginia K. Martin
PII:
S0020-1693(13)00120-5
http://dx.doi.org/10.1016/j.ica.2013.03.008
ICA 15366
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
12 December 2012
3 March 2013
8 March 2013
Please cite this article as: K.M. Williams, A.D. Poynter, J.D. Hendrie, D.C. Jackson, V.K. Martin, Comparison of
N-acetylmethionine reactivity between oxaliplatin and an oxaliplatin derivative with chiral (S,S) amine nitrogen
atoms, Inorganica Chimica Acta (2013), doi: http://dx.doi.org/10.1016/j.ica.2013.03.008
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1
Comparison of N-acetylmethionine reactivity between oxaliplatin and an oxaliplatin
derivative with chiral (S,S) amine nitrogen atoms
*
Kevin M. Williams , Amy D. Poynter, Jonathan D. Hendrie, Daniel C. Jackson, and Virginia K.
Martin
Department of Chemistry, Western Kentucky University, 1906 College Heights Blvd #11079,
Bowling Green, KY 42101-1079
*
Corresponding author: Kevin M. Williams, Department of Chemistry, Western Kentucky
University, 1906 College Heights Blvd #11079, Bowling Green, KY 42101-1079, Phone: 270-
45-8899, Fax: 270-745-5361, e-mail: kevin.williams@wku.edu
7

2
Abstract
We have synthesized an oxaliplatin derivative using N,N'-dimethyl-1,2-diaminocyclohexane
(
Me
2
dach) as the diamine ligand. The complex (S,R,R,S)-Pt(Me dach)(oxalate), where S,R,R,S
2
1
represents the chiralities at N,C,C,N, respectively, was prepared and characterized by H NMR
spectroscopy, COSY, NOESY, and HMQC. Oxaliplatin reacts with N-acetylmethionine (N-
AcMet) to form [Pt(dach)(N-AcMet-S) ] and [Pt(dach)(N-AcMet-S,N)], with the former favored at
2
higher molar ratios of N-AcMet. In contrast, Pt(Me dach)(oxalate) reacts to form
2
+
[
Pt(Me dach)(N-AcMet-S,O)] even in the presence of excess N-AcMet. Molecular mechanics
2
calculations are consistent with significant steric clashes in models of [Pt(Me dach)(N-AcMet-
2
S) ]. When N-AcMet was reacted with an excess of each platinum complex, the rate of N-
2
AcMet decrease was very similar for both complexes. Thus, the methyl groups at the nitrogen
atoms had little to no effect on the addition of the sulfur atom of a single N-acetylmethionine, but
they prevented chelation of the amide nitrogen or coordination of a second N-acetylmethionine
residue.
Key words
Nuclear magnetic resonance; platinum; oxaliplatin; amino acids; methionine
Abbreviations
dach, 1,2-diaminocyclohexane; Me dach, N,N'-dimethyl-1,2-diaminocyclohexane; N-AcMet, N-
2
acetylmethionine; ox, oxalate; Me en, N,N,N',N'-tetramethylethylenediamine; Et en, N,N-
4
2
5
diethylethylenediamine; Me dien, N,N,N',N',N"-pentamethyldiethylenetriamine
1. Introduction

3
The anticancer drugs cisplatin and oxaliplatin are known to interact with both DNA and
protein targets in vitro and in vivo. Whereas interaction with DNA is thought to result in the
cytotoxicity, interaction with amino acids and proteins could be involved in resistance
pathways,[1] oxidative stress,[2] and/or cellular uptake.[3] The amino acid methionine is a key
amino acid target, with small molecule studies indicating that reaction with methionine or related
thioether molecules is kinetically favored over reaction with guanine when the amine ligands are
relatively small.[4] Reactions of platinum(II) complexes with proteins may have additional
applications including their use as selective protein cleavage agents[5-7] or as specific enzyme
inhibitors[8, 9]. Thus, it is important to understand the factors that influence reactivity with
particular biologically relevant targets.
Addition of alkyl groups to the amine nitrogen atoms can affect the reaction of cisplatin
analogs with methionine. A platinum complex with the bulky triamine ligand N,N,N',N',N"-
pentamethyldiethylenetriamine (Me dien) reacted faster with 5'-GMP than with N-
5
acetylmethionine due to additional steric hindrance in reactions involving the latter.[10] More
2
+
recently, the coordination of one N-AcMet residue to [Pt(Me en)(D O) ] [Me en = N,N,N',N'-
4
2
2
4
tetramethylethylenediamine] was determined to be approximately 16-fold slower than
2
+
coordination of N-AcMet to [Pt(en)(D O) ] .[11] The significant slowing was attributed at least
2
2
in part to steric hindrance. By contrast, the rate of reaction with N-AcHis was not affected by the
additional size of the Me en ligand; thus, the size of the amine ligand could impact which amino
4
acid residues are preferentially targeted.
2
+
2+
Previously, it was found that [Pt(Me
4
en)(D
2
O)
2
]
and [Pt(Et
2
en)(D
2
O)
2
] [Et en = N,N-
2
diethylethylenediamine] reacted with only one Met or N-AcMet residue; chelates involving the
sulfur atom and a carboxylate oxygen atom of N-AcMet or the sulfur and nitrogen atoms of Met
were stable products.[12, 13] Upon reaction between the platinum complex and the methionine
sulfur atom, the remaining available coordination site would be cis to the methionine sulfur atom

4
and also cis to a bulky amine ligand, and thus steric hindrance prevented coordination of a
second methionine ligand or the chelation of the amide nitrogen of N-AcMet.
Oxaliplatin (Figure 1), a third generation platinum(II) anticancer drug, utilizes a (R,R)-1,2-
diaminocyclohexane (dach) ligand in addition to an oxalate leaving ligand. The rate constants
2
+
2+
for reaction with methionine were found to be similar for [Pt(en)(H
2
O)
2
] and [Pt(dach)(H
2
O) ]
2
complexes,[14] suggesting that the additional carbons of the dach ligand do not interfere with
coordination of an incoming methionine ligand. However, the stereochemistry of the carbons of
the dach ligand should limit the ligand flexibility, and such chirality has been previously found to
influence chirality introduced at the nitrogen atoms of diamine ligands[15, 16]. Thus, we
hypothesized that the stereochemistry of the dach ligand would influence the resulting chirality
at the nitrogen atoms when secondary amines were present.
In the current study, we have utilized commercially available N,N'-dimethyl-1,2-
diaminocyclohexane (Me dach) as a diamine ligand containing secondary amine nitrogen atoms
2
(Figure 1). This ligand is an analog of the dach ligand utilized in oxaliplatin, with one methyl
group added to each nitrogen atom for additional bulk that would be near the available
coordination sites. Since previous studies probing reaction at methionine[10-13, 17] have
generally utilized platinum(II) complexes with primary amine nitrogen atoms (en, dien) or tertiary
amine nitrogen atoms (Me en, Me dien), we were interested to see how the presence of a
4
5
secondary amine nitrogen atom influenced the reaction with methionine.
2. Materials and Methods
Silver nitrate (Sigma-Aldrich), oxalic acid (Acros), potassium tetrachloroplatinate (Sigma-
Aldrich), (R,R)-1,2-diaminocyclohexane (Sigma-Aldrich), (R,R)-N,N'-dimethyl-1,2-
diaminocyclohexane (Sigma-Aldrich), and N-acetylmethionine (Sigma-Aldrich) were used as
received.

5
2.1. Silver oxalate. Silver oxalate was prepared by a combination of 1 g of silver nitrate and 350
mg of oxalic acid in 20 mL of water. The reaction was stirred in an amber vial for ~30 minutes
and the insoluble silver oxalate was collected by filtration and washed with water.
2.2. Pt(dach)(ox) (oxaliplatin). In a typical reaction, 64 mg (0.56 mmol) of (R,R)-1,2-
diaminocyclohexane (dach) was dissolved in 5 mL of methanol and added dropwise to an
aqueous solution (5 mL) of 232 mg (0.56 mmol) of K PtCl . The reaction was stirred overnight
2
4
and a yellow precipitate of Pt(dach)Cl was collected and washed with water, ethanol, and ether.
2
Yield 128.8 mg (61%). Equimolar (0.17 mmol) amounts of Pt(dach)Cl and silver oxalate were
2
combined in ~35 mL of water in an amber vial and stirred overnight. The sample was filtered to
remove the AgCl precipitate and rotovapped with heating to ~35 °C to isolate Pt(dach)(ox).
1
Yield 27.9 mg (41%) H NMR (D O): 2.33 ppm (1 H, dd), 2.04 ppm (1 H, broad d), 1.56 ppm (1
2
H, m), 1.30 ppm (1 H, m), 1.15 ppm (1 H, m).
2
2.3. Pt(Me dach)(ox). In a typical reaction, 56.8 mg (0.4 mmol) of (R,R)-N,N'-dimethyl-1,2-
diaminocyclohexane (Me dach) in 5 mL methanol was added dropwise to an aqueous solution
2
(
5 mL) of 166 mg (0.4 mmol) K PtCl . After stirring overnight, a yellow precipitate was collected
2
4
by vacuum filtration (Pt(Me dach)Cl ) and washed with water, ethanol, and ether. Yield 100 mg
2
2
(
61%). An equimolar amount (75 mg) of silver oxalate was added to the Pt(Me dach)Cl and
2
2
the mixture was stirred in an amber vial for at least 24 hours. The sample was filtered to
2
+
remove AgCl precipitate to form Pt(Me dach)(ox) with some [Pt(Me dach)(H O) ] , which
2
2
2
2
converted to Pt(Me dach)(ox) when rotovapped with heating to ~35 °C. Yield: 72.9 mg (69%).
2
+
+
1
Mass spectrometry: 426.2 (M+H ), 448.3 (M+Na ). H NMR (D O): 2.53 ppm (3 H, s), 2.30 (1 H,
2
1
13
br), 2.27 (1 H, br), 1.62 (1 H, br), 1.12 (2 H, br). See Table 1 for H and C NMR assignments.
1
13
195
2
.4. NMR spectroscopy. H, C, and Pt NMR data were collected on a JEOL 500 MHz NMR
instrument. HMQC experiments utilized a J-coupling value of 140 Hz, and NOESY experiments

6
1
utilized a 500 ms mixing time. Spectra were referenced to the residual HOD signal ( H), TMS
1
3
195
(
C), or K PtCl ( Pt).
2
6
Stock solutions (typically 10 mM) of the appropriate platinum compound and N-
acetylmethionine were individually prepared in D O and the pH (uncorrected) adjusted to 4.
2
Aliquots of each individual solution were then combined in D
concentrations.
2
O to give the appropriate
2.5. Molecular mechanics. Molecular mechanics and dynamics calculations were performed as
described previously[13] using an AMBER force field modified to include parameters for
platinum[18] and methionine.[12] For each configuration and conformation, dynamics were
typically run for 250 ps at 300 K with structures saved every 1 ps; these structures were then
subjected to energy minimization.
3. Results
3
.1 Determination of stereochemistry of Pt(Me dach)(ox)
2
1
The H NMR spectrum of [Pt(Me
2
dach)(ox)] shows only one methyl group at 2.53 ppm
(
Table 1), indicating that only one diastereomer is formed and that it is C -symmetrical. Since
2
both chiral carbons have the R stereochemistry, the stereochemistry of the N atoms must be
both R or both S.
HMQC was used to definitively identify C1,2 and H1,2 since these were the only ring
carbons with only one hydrogen attached (see Figure 1 for numbering). The lack of a NOESY
or COSY cross peak of the H1,2 resonance to the signal at 1.62 ppm led to the assignment of the
.62 ppm signal as one of the H4,5 signals. The HMQC was then used to assign the remaining
1
13
H4,5 signal. The H , signals were assigned based on the remaining C signal in the HMQC
3 6
spectrum.

7
A NOESY spectrum acquired in DMSO-d
6
revealed a cross peak between the N-H
resonance at 6.38 ppm and one H3,6 resonance. No NOESY was observed between the N-H
resonance and the H1,2 resonances. Molecular mechanics models of (S,R,R,S)- and (R,R,R,R)-
Pt(Me dach)Cl revealed that the former would have ~2.4 Å between N-H and the axial H
2
2
3,6
whereas the latter would have a similar distance between the N-H and the H1,2 atoms. Thus,
the stereochemistry of the isomer was assigned to (S,R,R,S)-Pt(Me dach)(ox).
2
3.2 Characterization of the final products of the reaction of N-AcMet with
Pt(dach)(ox) and Pt(Me dach)(ox)
2
When N-AcMet and Pt(dach)(ox) were combined in a 2:1 ratio, broad resonances at
.01, 2.56, and 2.58 ppm were visible after 1 day (Figure 2); the 2.01 ppm signal is in an area
2
typical for Ac-CH
3
resonances of N-AcMet whereas the other two are characteristic of S-CH
3
signals in which the platinum atom has coordinated to the sulfur atom. When the sample was
heated, the broad resonances at 2.56 and 2.58 ppm coalesced into one signal and began to
1
95
sharpen (Figure 2). A Pt NMR spectrum showed a peak at -3739 ppm; this chemical shift is
in the -3600 to -3800 range that is seen for a PtN S coordination environment.[12, 19, 20] The
2
2
final product was therefore assigned to [Pt(dach)(N-AcMet-S)2].
When an equimolar solution of Pt(dach)(ox) and N-AcMet was monitored by NMR
spectroscopy, a resonance at 2.00 ppm and a broad resonance at 2.34 ppm, just downfield of
the H1,2 resonance of oxaliplatin at 2.33 ppm, grew over the first 2 h but eventually disappeared
by 24 h (Figure 3). These resonances were assigned to the intermediate [Pt(dach)(N-AcMet-
-
S)(ox-O)] . The dominant products after 24 hours had S-CH
3
resonances at 2.30, 2.37 (two
nearly overlapping resonances), and 2.43 ppm and overlapping Ac-CH resonances at 2.01
3
ppm. The shifts of these S-CH resonances are very similar to those observed previously for
3
[Pt(en)(N-AcMet-S,N)] complexes; four products were possible due to chirality at the S atom

8
and the possibility of cis/trans isomers around the amide bond.[17] Thus, we assign the major
products of the equimolar Pt(dach)(ox) reaction with N-AcMet to the isomers of [Pt(dach)(N-
AcMet-S,N)], with the platinum coordinated to the sulfur atom and the (deprotonated) amide
1
95
nitrogen atom. The Pt NMR spectrum showed signals at -3215 and -3260, which are in the
range expected for a PtN S coordination environment.[19, 20] Small broad signals at 2.56 and
3
1
2
.58 ppm in the H NMR spectrum were also observed, and these sharpened with the
temperature was increased; thus some [Pt(dach)(N-AcMet-S) ] also formed in this reaction.
2
The final NMR spectrum of the reaction of N-AcMet with Pt(Me dach)(ox) showed three
2
doublet of doublets in the range of 5.7-5.9 ppm (Figure 4) regardless of the molar ratios of the
N-AcMet or platinum complex. These signals are characteristic of the α-hydrogen of N-AcMet
residues chelated via the sulfur and carboxyl oxygen atoms[12, 13]; HMQC results showed a
1
3
corresponding C chemical shift of 54.8 ppm, consistent with the α-carbon of N-AcMet[17]. The
1
95
Pt NMR spectrum showed peaks at ca. -2870 and -2890 ppm. This is within the range of -
2
600 to -2900 ppm that is typical for a PtN SO coordination environment[12, 13] but is downfield
2
of the typical range for the PtN S coordination environment (-3000 to -3300 ppm) and
3
significantly downfield of the observed shifts for [Pt(dach)(N-AcMet-S,N)]. The significant
1
95
downfield shift of the H signals and the Pt NMR shift indicate that the products are
α
+
assignable to [Pt(Me dach)(N-AcMet-S,O)] .
2
When the temperature was raised, several of the resonances broadened, indicating
multiple species are in chemical exchange (Figure 5). A similar broadening was noted for
+
[
Pt(Et
2
en)(N-AcMet-S,O)] previously[13] and was attributed to the differences in the R and S
chiralities at the sulfur atom upon coordination to the platinum.
3.3 Molecular mechanics calculations

9
Previously, molecular mechanics calculations have been used to model [Pt(Me
4
en)(9-
EtG) ] and [Pt(Me en)(Met) ] complexes in order to understand better why the latter complexes
2
4
2
do not form in solution[12]. The minimum energy structure of the latter was >21 kcal/mol higher
in energy than the minimum energy of the former; by comparison, the minimum energy of cis-
[Pt(NH
3
)
2
(Met)
2
] was only 7 kcal/mol higher than the minimum energy of cis-[Pt(NH
3
)
2
(9-EtG) ].
2
In the present study, we determined the minimum energy of various conformations of
Pt(dach)(9-EtG) ], [Pt(dach)(Met) ], [Pt(Me dach)(9-EtG) ], and [Pt(Me dach)(Met) ] (Table 2).
[
2
2
2
2
2
2
The methionine complex was <6 kcal/mol higher in energy than the guanine complex when the
dach ligand was utilized, which is comparable to the calculated difference between analogous
complexes with cisplatin. In contrast, the methionine complex was >17 kcal/mol higher in
energy for complexes having the Me dach ligand; this value is closer to the ~21 kcal/mol seen
2
for the Me en ligand previously. The structures of [Pt(Me dach)(Met) ] had significant distortion
4
2
2
of the platinum coordination plane due to steric clashes (Supplementary Material).
+
Molecular mechanics calculations were also performed on [Pt(Me dach)(Met-S,O)] with
2
either the R or S chirality at the sulfur atom and a variety of conformations of the 7-membered
chelate ring. Overall, we found three structures that were significantly lower in energy (by more
than 4 kcal/mol) than other structures and that had relatively few steric clashes (Supplementary
material). Thus, the observation of three sulfur, oxygen chelates in the molecular mechanics is
consistent with the experimental observation of three sets of NMR resonances for
+
[
Pt(Me dach)(N-AcMet-S,O)] .
2
3
.4 Comparison of the reactivity of Pt(dach)(ox) and Pt(Me dach)(ox) with N-AcMet
2
In order to compare the relative reaction rates of Pt(dach)(ox) and Pt(Me dach)(ox) with
2
N-AcMet, we used 5 mM concentrations of each platinum complex and 1 mM concentrations of
N-AcMet; these conditions favor coordination of only one N-AcMet to Pt(dach)(ox). Because

1
0
both mono products and chelates are possible in each reaction and the signals from the
unreacted platinum interfere with many of the key product signals, the reaction rates were
monitored primarily by observing the decrease in the free N-AcMet signals. Both Pt(dach)(ox)
and Pt(Me dach)(ox) react with a half-life of approximately 1 hour under these conditions
2
(Figure 6). By 4 hours, the unreacted N-AcMet was nearly depleted in both samples. In
2
+
contrast, [Pt(Me en)(D O) ] reacted ~16 times more slowly with N-AcMet than did
4
2
2
2
+
[
Pt(en)(D O) ] .[11] Thus, the presence of a secondary amine N cis to the coordination site in
2
2
Pt(Me dach)(ox) did not significantly affect the rate of the first N-AcMet coordination.
2
4. Discussion
The (R,R)-N,N'-dimethyl-1,2-diaminocyclohexane (Me dach) ligand is commercially
2
available and coordinates to platinum to produce one diastereomer of Pt(Me dach)(ox), with the
2
stereochemistry of (S,R,R,S) at the N,C,C,N atoms, respectively. Chirality in the carbon atoms
of a ring system has previously been shown to influence the nitrogen stereochemistry[15, 16].
The use of the Me dach ligand leads to a relatively simple synthesis of a platinum(II) diamine
2
complex with a C -symmetrical ligand having chirality at the nitrogen atoms.
2
A platinum(II) triamine complex with the bulky Me dien ligand was previously shown to
5
slow reaction of N-acetylmethionine dramatically relative to 5'-GMP, and thus we speculated
that large amine ligands on platinum(II) complexes could lead to less reaction with proteins
relative to DNA.[10] The Me dien ligand represents an extreme case, with tertiary amines
5
present at both coordination sites cis to the available coordination site. Previously, a
platinum(II) complex with the DNSH-dien ligand, which has a primary amine and a bulky
sulfonamido ligand cis to the available coordination site, was found to react with 5'-GMP and
Met at roughly equal rates.[21] More recently, research on phenanthriplatin, a platinum(II)
complex with a bulky anthracene-like ligand cis to the available coordination site found that

1
1
reaction with N-AcMet and 5'-dGMP occurred at similar rates; this platinum(II) complex had
higher activity than less bulky monofunctional complexes such as pyriplatin, and reduced
reaction with proteins relative to DNA was proposed as a possible reason.[22]
When excess N-AcMet is present, the final product of the reaction with Pt(dach)(ox) is
[
Pt(dach)(N-AcMet-S)
2
] (Figure 7, left). When excess or equimolar Pt(dach)(ox) is present,
[Pt(dach)(N-AcMet-S,N)] is the dominant final product (Figure 7, middle). Previously, reaction of
Pt(dach)(ox) with Met has also been shown to result in a S,N chelate.[23] Also, [Pt(en)(N-
AcMet-S,N)] has been an observed product in reactions with N-AcMet.[17]
Several reports have indicated that Pt(dach)(ox) reacts with Met to form [Pt(dach)(Met-
S,N)] as a major species[23-25] even in the presence of excess methionine[23]. However, N-
AcMet forms some [Pt(dach)(N-AcMet-S) ] even at equimolar ratios (Figure 3) and as a
2
dominant product when excess N-AcMet is present (Figure 2). The amine nitrogen of Met
would be expected to be a better ligand than the amide nitrogen of N-AcMet; thus, the
observation of the S,N chelate in the presence of excess Met may be due to a faster chelation
of the amine nitrogen compared with the amide nitrogen in N-AcMet .
The presence of a tertiary amine nitrogen cis to the available coordination site prevented
coordination of a second methionine or chelation of the amide nitrogen of N-AcMet to
complexes utilizing the Me en or Et en ligands; steric clashes between the acetyl group or the
4
2
second methionine and the methyl or ethyl groups of the amine ligand were significant.[12, 13]
Thus, chelates involving the sulfur atom and the carboxyl oxygen atom were observed. In the
+
present study, [Pt(Me dach)(N-AcMet-S,O)] (Figure 7, right) is a stable product even in the
2
presence of excess N-AcMet. Therefore, the secondary amine atoms of the Me dach also
2
cause significant steric clashes to prevent coordination of the amide nitrogen and/or second
methionine residue. Molecular mechanics calculations (Table 2) support the presence of more

1
2
severe steric clashes between methionine residues and the Me dach ligand compared with the
2
dach ligand. Our previous studies[11-13] establish that coordination of a methionine trans to a
tertiary amine nitrogen atom effectively blocks coordination of a second methionine residue cis
to the methionine. Our current studies indicate that coordination trans to a secondary amine
nitrogen also prevents the second methionine residue from reacting. A recent study found that
a modified dach ligand with a 2-butyl substituent on one of the nitrogen atoms of an oxaliplatin
derivative led to a relatively high antitumor activity and low toxicity for the platinum(II)
complex.[26] Agarose gel electrophoresis experiments suggested that the complex could be
binding to the DNA in a monofunctional fashion. Thus, the presence of a large substituent on a
secondary amine could affect DNA reactivity.
1
Three different S,O products were observed by H NMR spectroscopy, and the signals
from these complexes broadened at higher temperature, indicating chemical exchange. Two
different chiralities at the sulfur atom would lead to diastereomers that could be distinguished by
NMR spectroscopy; additionally, several possible conformations of the 7-membered chelate ring
are possible, and interconversion of these conformations could be slow due to the presence of
the methyl groups on the Me dach ligand. Our molecular mechanics calculations showed three
2
structures that were relatively low in energy and free of steric clashes.
The presence of one methyl group on each N is sufficient to prevent coordination of a
second N-AcMet (Figure 4). However, the rate of coordination of the first methionine does not
appear to be affected, as Pt(dach)(ox) and Pt(Me dach)(ox) reacted with N-AcMet at similar
2
2
+
rates (Figure 6). By contrast, [Pt(Me
4
en)(D
2
O)
2
] reacted with N-AcMet approximately 16 times
2
+
slower than did [Pt(en)(D
2
O)
2
] .[11] Thus, whereas ligands such as Me
4
en and Me dien would
5
be expected to reduce the reactivity of methionine relative to other protein or DNA targets, the
Me dach ligand would not. The Me dach ligand thus is unique in that the additional methyl
2
2
groups affect coordination of the second coordination site but not of the first coordination site.

1
3
Acknowledgement
This research was supported by awards from the NIH (1R15 GM074663-01) and
Western Kentucky University (Research and Creative Activities Program RCAP 11-8042) to
KMW. KMW and DCJ were also supported by a Faculty Undergraduate Student Engagement
grant from Western Kentucky University (12-SF145). JDH was supported as part of a Research
Experience for Undergraduates award from the National Science Foundation (DBI-1004665).
Appendix A. Supplementary Material
Stereo renderings of minimum energy molecular mechanics structures of [Pt(dach)(9-
EtG)
2
], [Pt(dach)(Met)
2
], [Pt(Me
2
dach)(9-EtG)
2
], [Pt(Me
2
dach)(Met) ], and the three lowest
2
energy structures of [Pt(Me dach)(Met-S,O)].
2

1
4
References
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1
5
List of Figures
Figure 1. Representations of (R,R)-Pt(dach)(ox) [oxaliplatin] and (S,R,R,S)-Pt(Me dach)(ox).
2
1
Figure 2. Partial H NMR spectra of 3.33 mM Pt(dach)(ox) and 6.67 mM N-AcMet. Each
spectrum has the y-axis scaled for the largest peak. The * indicates the S-CH
product.
3
signals of the
1
Figure 3. Partial H NMR spectra of 3.33 mM Pt(dach)(ox) and 3.33 mM N-AcMet. Each
spectrum has the y-axis scaled for the largest peak. The * indicates the S-CH
chelates.
3
signals of the
1
Figure 4. Partial H NMR spectrum of 10 mM Pt(Me dach)(ox) and 10 mM N-AcMet. Each side
2
of the spectrum has the y-axis scaled for the largest peak.
1
+
Figure 5. Partial H NMR spectra of [Pt(Me dach)(N-AcMet-S,O)] at several temperatures. All
2
spectra are referenced to the HOD signal adjusted for temperature.
1
Figure 6. Partial H NMR spectra of 5 mM Pt(dach)(ox) (top) or Pt(Me dach)(ox) (bottom)
2
reacted with 1 mM N-AcMet. For a given platinum complex, the spectra at each time point are
scaled by the same factor. The unreacted N-AcMet signals are the two singlets of
approximately equal size at ~2.0 and 2.1 ppm indicated by * in the spectra at 15 min. In the top
spectrum, the broad peaks seen in all spectra are from the dach ligand of unreacted
Pt(dach)(ox) and the large singlet increasing in size over time is the acetyl CH signal of
3
product. The broad signals in the bottom spectra are acetyl CH product signals.
3
Figure 7. Representations of [Pt(dach)(N-AcMet-S)
2
], [Pt(dach)(N-AcMet-S,N)], and
+
[Pt(Me
2
dach)(N-AcMet-S,O)] complexes, respectively.

1
6
1
13
Table 1. Assignments of H and C NMR resonances of Pt(Me
2
dach)(ox) in D O. All values in
2
ppm.
C-H1,2
2.27
C-H3,6
1.12 (ax)
C-H4,5
N-CH3
2.53
1
1
H
1.12 (ax)
1.62 (eq)
24.39
2.30 (eq)
3
C
70.83
28.23
38.68

1
7
Table 2. Calculated minimum energies of [PtA
2
(9-EtG)
en. All energies in kcal/mol.
2
] and [PtA
2
(Met)
2
] complexes, where A
2
=
dach, Me
2
dach, cis-Pt(NH
3
)
2
, or Me
4
Pt ligand
dach
PtA G energy
PtA Met energy
Difference
5.5
17.2
7.1
21.5
2
-17.0
-11.7
-17.7
-5.4
2
2
-11.5
5.5
2
Me
2
dach
a
cis-Pt(NH
3
)
2
-10.6
16.1
a
Me en
4
a
Reference [12]

1
8
Graphical abstract
Oxaliplatin and an analog with chiral secondary amine nitrogen atoms were each reacted with N-
acetylmethionine. Whereas the reaction at the first coordination position was unaffected by the
size of the amine ligand, the analog was unable to coordinate a second methionine or chelate the
amide nitrogen.

Figure 1
NH2
NH2
Pt
N
N
6
3
5
4
1
2
Pt
CH3

Figure 2
1
day
*
*
1
day, 50 C
2.5
2.0

Figure 3
2
h
1
day
*
*
*
2
2
days, 50 C
*
*
*
.5
2.0

Figure 4
5.8 5.7
2.7
2.0

Figure 5
8
0 C
0 C
6
40 C
5.9
5.7
2.7
2.4
2.1 2.0

Figure 6
*
*
dach
*
15 min
1 h
2 h
4 h
*
Me dach
2
2.1
2.0
2.1
2.0
2.1
2.0
2.1
2.0

Figure 7
H
N
H N
NH2
S
H2N
S
NH2
N
H
2
Pt
Pt
O
Pt
N
O
S
S
O–
O
O
O
O
O
HN
N
H
O
O–
HN
O–
O

1
9
Highlights
•
•
•
•
•
Formed Pt(Me
2
dach)(oxalate), where Me dach = N,N'-dimethyl-1,2-diaminocyclohexane
2
Stereochemistry determined to be (S,R,R,S) for Pt(Me dach)(ox)
Pt(dach)(ox) and Pt(Me dach)(ox) react with N-acetylmethionine at similar rates
Pt(dach)(ox) can form [Pt(dach)(N-AcMet-S)
2
2
2
] or [Pt(dach)(N-AcMet-S,N)]
+
Pt(Me dach)(ox) can form only [Pt(Me dach)(N-AcMet-S,O)]
2
2

Graphical abstract
H
N
H N
NH2
S
N
H
2
Pt
Pt
S
S
O
O
O
O
O
HN
N
O
O–
H
HN
O–
O