Synthesis of Mitomycin C and Decarbamoylmitomycin C N2 deoxyguanosine-adducts

Article abstract of DOI:10.1016/j.bioorg.2016.02.003

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) - a derivative of MC lacking the carbamate on C10 - are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-lin

Full text of DOI:10.1016/j.bioorg.2016.02.003

Bioorganic Chemistry 65 (2016) 90–99
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of Mitomycin C and Decarbamoylmitomycin C N2
deoxyguanosine-adducts
a,b,
a
a
a
Elise Champeil a,b, , Shu-Yuan Cheng
, Bik Tzu Huang , Marta Conchero-Guisan , Thibaut Martinez ,
⇑
⇑
Manuel M. Paz c, Anne-Marie Sapse a
a John Jay College of Criminal Justice, New York, 524 West 59th Street, New York, NY 10019, USA
b The Graduate Center of the City University of New York, New York, NY 10016, USA
c Departamento de Química Orgánica, Facultade de Química, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) – a derivative of MC lacking the carbamate on
C10 – are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA mono-
adducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by
MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine–mitosene bond:
trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure
from trans or cis adducts are responsible for the different biochemical responses produced by MC and
DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypoth-
esis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However,
this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mito-
sene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononu-
cleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine
with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional
Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or
bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations
showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for
MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the acti-
vated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are pos-
sible in a cellular environment.
Received 24 November 2015
Revised 19 January 2016
Accepted 11 February 2016
Available online 11 February 2016
Keywords:
Mitomycin C
Deoxyguanosine
Post-oligomerization
Stereo preference
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
generated by treatment of EMT6 mouse mammary cells with MC or
DMC have opposite stereochemistry at carbon one of the guanine–
Mitomycin C (MC) [1] is a DNA alkylating agent [2] currently
used to treat certain stomach, anal and lung cancers [3]. Its cyto-
toxicity is attributed to the formation of DNA-adducts, in particular
to the formation of interstrand crosslinks (ICLs) [4]. 10-
decarbamoylmitomycin C (DMC) is a derivative of MC lacking the
carbamate group on C-10 (Fig. 1). As such, DMC was originally
thought to only alkylate DNA monofunctionally. However, when
EMT6 mouse mammary tumor cells were treated with DMC, both
ICLs and DNA monoadducts were produced [4]. Major DNA adducts
mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC
[4].
In correlation with the stereopreference exhibited by DMC, it
was found that DMC was more toxic to human cancer cells and
that only DMC induces apoptosis efficiently in a p53 independent
manner [5]. Access to mitosene-alpha and mitosene-beta DNA-
adducts is essential to elucidate if and how the local DNA structure
of these adducts is responsible for the different biochemical
responses displayed by the two drugs. Individual structure–activ-
ity relationships of multiple DNA adducts generated by a single
agent have been investigated mostly in the case of organic muta-
gens and carcinogens [6]. In general, such studies utilize synthetic
oligonucleotides bearing a specific adduct at a unique position of
their base sequences [7]. Similar investigations can be conducted
on MC and DMC adducts, enabling direct comparisons of biological
⇑
Corresponding authors at: John Jay College of Criminal Justice, New York, 524
west 59th Street, New York, NY 10019, USA.
E-mail addresses: echampeil@jjay.cuny.edu (E. Champeil), shcheng@jjay.cuny.
edu (S.-Y. Cheng), biktzu.huang@jjay.cuny.edu (B.T. Huang), mconcheiro-guisan@
jjay.cuny.edu (M. Conchero-Guisan), thibaut.martinez@etu.chimie-paristech.fr
(T. Martinez), manuel.paz@usc.es (M.M. Paz), acransg6@aol.com (A.-M. Sapse).
http://dx.doi.org/10.1016/j.bioorg.2016.02.003
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

E. Champeil et al. / Bioorganic Chemistry 65 (2016) 90–99
91
effects induced by a different stereochemistry at carbon 1 (Fig. 1).
2. Materials and methods
Trans adducts (or alpha) are available by reacting oligonucleotides
with activated MC (bio-mimetic synthesis) [8], however, to this
day, no site specific cis (or beta) adduct has been synthesized.
Examination of the biochemical, structural, and biological effects
of beta adducts has been severely limited by the inability to pre-
pare sufficient quantities of this lesion. Our long term goal is to
synthesize oligonucleotides bearing the beta ICL using a post-
oligomerization strategy. Previously, we presented the synthesis
of major Mitomycin C-DNA adducts both at the nucleoside [9,10]
and at the oligonucleotide level [10] using post-oligomerization.
This report presents the first chemical synthesis of a mitosene beta
(cis) monoadduct; which is the first step toward the post-
oligomerization synthesis of site specific beta ICLs.
MC and DMC must be reduced in a monofunctional or bifunc-
tional way before alkylating DNA [11]. In cells, the major mode
of activation is bifunctional [11]. In this case, DMC produces mostly
beta adducts and MC alpha adducts. A rationale for the opposite
stereochemistry displayed by MC and DMC at CpG steps in DNA
has been suggested [12]. On the other hand, the major adduct
formed in the reaction of DMC with calf thymus DNA or poly(dG.
dC)poly(dG.dC) is the alpha monoadduct, contrary to what hap-
pens in a cellular environment [4]. This apparent contradiction
warrants some investigation. Factors responsible for the stereo-
preference exhibited by MC and DMC toward deoxyguanosine
independently of the DNA structure have not been determined
yet. Therefore, we also present here a study on the reactivity of
deoxyguanosine with MC and DMC in an effort to understand the
factors leading to the favored stereoselectivity. Different types of
drug activation (monofunctional and bifunctional) have been con-
sidered along with different solvent conditions. Molecular calcula-
tions have also been performed to measure the energetics of all
reactions examined.
2.1. General information
1H NMR and 13C NMR spectra were recorded using a JEOL ECX
300 (300 MHz), a Varian Inova 500 (500 MHz) or a Bruker AVANCE
500 (500 MHz) spectrometer. Spectra were recorded at 298 K and
the residual solvent peak was used as the internal reference. Chem-
ical shifts are reported in parts per million and coupling constants
are in hertz (Hz). The conventional numbering system is used for
the mitosene moiety and the purine carbons are numbered 1–6
also as per convention. The sugar carbons are numbered 10–50
beginning at the anomeric carbon and proceeding via the carbon
chain to the primary carbinol center. Mitosene compounds with
trans substituents at carbon 1 and 2 are labeled ‘‘a” and those with
cis substituents at carbon 1 and 2 are labeled ‘‘b”.
Reagents were obtained from commercial sources and were
used without further purification. All reactions were carried out
under an atmosphere of Argon unless otherwise stated. Thin layer
chromatographic analyses were carried out on 250 mm silica gel
plates containing a fluorescent indicator and, if necessary, spots
were visualized with I2. Column chromatographic purifications
were performed using 200–300 mesh silica gel. Trans-2,7-dia
mino-1-hydroxymitosene
and
cis-2,7-diamino-1-
hydroxymitosene were synthesized by known methods [13]. Com-
pounds 6a and 6b were synthesized according to a previously
described procedure [9]. Adducts 1a, 1b, 2a and 2b generated via
direct alkylation were isolated by chromatography using an Agilent
1200 HPLC system equipped with
a Kromasil C18 column
(4.6 mm ⁄ 250 mm). Ratios of cis and trans adducts were calcu-
lated using the relative absorption of each adduct at k = 254 nm.
HRMS spectra were recorded by direct infusion using Bruker’s
micrOTOF-II ESI instrument at Notre Dame University Mass Spec-
Fig. 1. Major DNA adducts generated by MC and DMC in cells.

92
E. Champeil et al. / Bioorganic Chemistry 65 (2016) 90–99
trometry Facility. LC-ESI MS/MS experiments were performed at
John Jay College using a Shimadzu LC-MS 8030 (Shimadzu Corpo-
ration, Kyoto, Japan). CD spectra were obtained with a Jasco 1500
CD spectrometer. UV spectra were obtained using a Shimadzu UV
2600 UV–vis spectrometer.
OasisÓ HLB, 12 cc, 500 mg; 15% acetonitrile in water). Structure of
1b was confirmed by UV and CD spectroscopy (supporting infor-
mation Figs. S13 and S14) and co-injection with a sample obtained
via the bio-mimetic route. HRMS m/z calcd for C24H28N9O8 [M+H]+:
570.2055, found: 570.2029.
2.5. HPLC conditions for isolation of Mitomycin C N2 deoxyguanosine
adducts (1a and 1b) and Decarbamoylmitomycin C N2
deoxyguanosine adducts (2a and 2b) from direct alkylation reactions
2.2. Synthesis of 7b
Compound 6b (33 mg, 0.062 mmol) was dissolved in pyridine
(3 mL) and ammonium hydroxide (1.5 mL). Trimethylphosphine
(69 lL, 0.67 mmol) was added. The reaction was stirred at room
Analysis of MC and DMC adducts (1a, 1b, 2a, 2b) was performed
using the following HPLC system: Buffer A: ammonium acetate;
20 mM, pH 5.5; Buffer B: acetonitrile. Flow: 1 mL/min. For MC
adducts 1a and 1b: Gradient: from 0 min to 45 min, isocratic 5%
B; from 45 min to 55 min, 5–8% B; from 55 min to 90 min, 8–11%
B. Adducts appear at RT = 56 min for 1a and RT = 76 min for 1b.
For DMC adducts 2a and 2b: Gradient: from 0 min to 45 min, iso-
cratic 6% B; from 45 min to 55 min, 6–9% B; from 55 min to
70 min, isocratic 9% B. Adducts appear at RT = 30 min for 2a and
RT = 64 min for 2b. Examples of chromatograms can be seen in
the supporting information (Figs. S1 and S2).
temperature in a sealed vial flushed with Argon for 30 min. Sol-
vents were evaporated and the red residue was purified by prepar-
ative thin layer chromatography (tlc) (SiO2: 1% NH4OH: 10%
MeOH: 89% CH2Cl2) yielding 29 mg of 6b (93% yield). 1H NMR
(methanol-d4) d = 0.09 (s, CH3, 9H), 1.05 (t, CH2, J = 8.3 Hz, 2H),
1.82 (s, CH3, 3H), 4.00 (dd, H3, J = 8.2, 12.5 Hz, 1H), 4.22 (t, CH2,
J = 8.3 Hz, 2H), 4.46 (d, H1, J = 5.4 Hz, 1H), 4.54 (dd, H3, J = 8.3,
12.2 Hz, 1H), 4.70 (m, H2, 1H), 5.23 and 5.30 (AB quartet, CH2,
H
10, J = 12.7 Hz, 2H). 13C NMR (methanol-d4) d = ꢀ1.4 (3C), 8.3
(1C), 18.6 (1C), 55.2 (1C), 57.4 (1C), 58.4 (1C), 64.2 (1C), 66.2
(1C), 106.4 (1C), 114.2 (1C), 123.5 (1C), 129.6 (1C), 141.1 (1C),
148.6 (1C), 154.3 (1C), 159.2 (1C), 178.5 (1C), 179.6 (1C). HRMS
m/z calcd for C20H30N5O6Si [M+H]+: 464.1965, found: 464.1958.
2.6. Autocatalytic or bifunctional activation of Mitomycin C and
decarbamoyl mitomycin C in buffer
2.3. Synthesis of 9b
Potassium phosphate buffer (autocatalytic conditions: 34 mL,
0.01 M, pH 7.5 or bifunctional conditions: 34 mL, 0.01 M, pH 5.8)
was deaerated 15 min with Argon. 20-deoxyguanosine (336 mg,
2-Fluoro-O6-(2-p-nitrophenylethyl)deoxyinosine
0.064 mmol) was dissolved in dry dimethylsulfoxide (200
Compound 7b (15 mg, 0.032 mmol) and diisopropylethylamine
(10 L, 0.96 mmol) were added to the reaction mixture which
(27 mg,
L).
l
1.36 mmol) was added with either MC (90 mg, 269
DMC (78 mg, 269 mol). The reaction mixture was stirred and
deaerated for 30 min. Sodium dithionite (autocatalytic conditions:
1.79 mL of a 40 mM solution, 72 mol, 0.27 equivalent or bifunc-
tional conditions: 10.1 mL of a 40 mM solution, 403.5 mol, 1.5
lmol) or
l
l
was incubated at 45° for three weeks. The resulting crude material
was dried with a flow of Argon to remove all solvents and further
removal of trace of dimethylsulfoxide was performed under high
vacuum. The desired product was isolated by preparative tlc
(SiO2: 15% methanol: 6% NH4OH: 79% CH2Cl2) to give 15 mg (54%
yield) of 9b (NMR spectrum shown in supporting information
Fig. S12).
l
l
equivalent) in deaerated potassium phosphate buffer (autocat-
alytic conditions: 0.01 M, pH 7.5 or bifunctional conditions:
0.01 M, pH 5.8) was added in one portion to the reaction mixture.
After stirring for one hour at room temperature under Argon, the
reaction mixture was opened to air and stirred for another
30 min. The reaction mixture was then centrifuged (13,000 rpm,
10 min) and prepurified using C18 chromatography (sep-pak
OasisÓ HLB, 12 cc, 500 mg; 5–20% acetonitrile in water). Adducts
were isolated in the 15% acetonitrile fraction. Further purification
was performed by HPLC.
1H NMR (methanol-d4): d ꢀ0.10 (s, CH3, 9H), 0.70 (t, CH2,
0
00
J = 8.3 Hz, 2H), 1.84 (s, CH3, 3H), 2.38 (m, H2 , 1H), 2.82 (m, H2
,
0
00
1H), 3.75 (dd, H5 , J = 12, 4 Hz, 1H), 3.82 (dd, H5 , J = 12, 3 Hz, 1H),
0
3.98 (m, H4 and CH2, 3H), 4.14 (dd, H3, J = 13, 7 Hz, 1H), 4.55 (m,
0
H3 , 1H), 4.58 (dd, H3, J = 13, 7 Hz, 1H), 4.74 (m, CH2, 2H), 5.05
(m, H2, 1H), 5.07 and 5.10 (AB quartet, CH2, H10, J = 7 Hz, 2H),
0
5.75 (d, H1, J = 6 Hz, 1H), 6.32 (t, H1 , J = 7 Hz, 1H), 7.62 (d, Har,
J = 8 Hz, 2H), 8.10 (s, H8, 1H), 8.17 (d, Har, J = 8 Hz, 2H), 8.55 (s,
NH, 1H). HRMS m/z calcd for C38H47N10O12Si [M+H]+: 865.3139,
found: 863.3147.
2.7. Autocatalytic or bifunctional activation of Mitomycin C and
decarbamoyl mitomycin C in DMSO
DMSO (1.88 mL) was first deaerated with Argon during 15 min.
2.4. Synthesis of 1b
20-deoxyguanosine (150 mg, 562
l
mol), MC (5 mg, 15
mol) was then added. The reaction mixture
was stirred and deaerated during 30 min. Sodium dithionite (auto-
catalytic conditions: 10 L of a 400 mM solution, 4 mol, 0.27 eq.
or bifunctional conditions: 56.2 of 400 mM solution,
mol, 1.5 eq or autocatalytic/bifunctional conditions:
L of a 400 mM solution, 15 mol, 1 eq) was added in one
mol, 2.58 mg)
lmol) or
DMC (4.3 mg, 15
l
The N2- and O6-protected mitosene–nucleoside adduct 9b
(12.0 mg, 0.017 mmol) was dissolved in dimethylsulfoxide
l
l
(50
(10
l
l
L) and was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene
L, 0.06 mmol). All starting material disappeared after 2 h.
l
L
a
22.5
37.5
l
l
The resulting crude material was diluted with water (1 mL) and
lyophilized. The product was isolated by preparative tlc (SiO2: 3%
NH4OH: 15% MeOH: 82% CHCl3) to give the N2-protected mito-
sene–nucleoside adduct 10b. Deprotection at the O6 position was
confirmed by HRMS analysis: m/z calcd for C30H40N9O10Si [M
+H]+: 714.2662, found: 714.2641. The intermediate 10b was
deprotected at the N2 position using tBuN4F (tetrabutylammonium
l
portion. P-Toluenesulfonic acid (1 equivalent, 15
l
from a 80 mM solution in deaerated DMSO was added immediately
afterward. After stirring for one hour at room temperature under
Argon, the reaction mixture was opened to air and stirred for
another 30 min. Solvents were evaporated and the residue was dis-
solved in water (1 mL). The solution was centrifuged (13,000 rpm,
10 min) and prepurified using C18 chromatography (sep-pak
OasisÓ HLB, 12 cc, 500 mg; 5–20% acetonitrile in water). Adducts
were isolated in the 15% acetonitrile fraction. Further purification
was performed by HPLC.
fluoride, 1 M THF solution, 0.68 mmol, 68
TAS-F (trisulfonium difluorotrimethylsilicate, 0.68 mmol, 18.7 mg)
in 50 L of DMF. Compound 1b (6.2 mg, 64% yield) was isolated by
precipitation in hexane followed by C18 chromatography (sep-pak
lL) in 50 lL of DMSO or
l

E. Champeil et al. / Bioorganic Chemistry 65 (2016) 90–99
93
2.8. Yield of direct alkylation reactions
2.10. Density Functional Theory (DFT) calculations
Estimated yields of direct alkylation reactions were obtained
through HPLC analysis of crude reaction mixtures. A 5 point cali-
bration curve was built where the area of the absorption at
312 nm of the mitosene chromophore (X axis) was plotted versus
the amount of mitosene chromophore (Y axis) – supporting infor-
mation, Fig. S11 [14]. For each reaction, a portion of the crude mix-
ture was analyzed and the peak area from alpha and beta adducts
was measured at 312 nm. The calibration curve was used to deter-
mine the quantity of adduct per injection.
Mitomycin C, Decarbamoylmitomycin C and their activated
species were studied with quantum mechanics, specifically the
Density Functional Theory (DFT), using the B3LP method, with
the 6-31 G⁄⁄ basis set, as implemented by the Spartan program
[16]. The energies of the different entities are obtained by geome-
try optimization in order to obtain global minima and the results
are displayed in Table S2, supporting information. Optimized fig-
ures are shown in the supporting information section (Figs. S16–
S31).
3. Results and discussion
2.9. Characterization of the MC and DMC adducts
3.1. Chemical synthesis of a mitosene-deoxyguanosine beta adduct
All four adducts (1a, 1b, 2a, 2b) were isolated by HPLC and then
characterized by LC-MS using a modified published procedure [15].
The column used was a Phenomenex Luna 3 lm C8, 100 Å,
Direct alkylation reactions between MC/DMC and deoxyguano-
sine are not efficient methods to synthesize nucleoside or nucleo-
tide adducts. At best, alpha adducts can be obtained in short
oligonucleotides via a bio-mimetic route, but the yield of adducts
varies with the length and the sequence of the nucleotide [17,8].
Our lab has been involved in alternative methods to access these
DNA adducts. We successfully synthesized adduct 1a and a 2,7-
150 mm ꢁ 2 mm. The LC column was maintained at 35 °C. Mobile
phase (MP) conditions were: MP A, 5 mM ammonium formate in
water containing 0.1% formic acid, and MP B, acetonitrile. Elution
gradient: 95% A for 15 min, 5–30% B in 30 min, 30% B for 15 min,
back to initial gradient conditions at min 61 and column equilibra-
tion for 4.5 min. The flow rate was 0.2 mL/min. The mass spec-
trometer (MS) was operating with dual ion source (DUIS) in
positive mode and data was collected in SIM mode. The MS param-
eters were: interface voltage 4.5 kV, nebulizer gas 2 L/min, desol-
vation line temperature 250 °C, heat block temperature 400 °C
and drying gas flow 15 L/min. The m/z monitored for adducts 1a
and 1b was 571, and for 2a and 2b was 528.
The chromatogram for each compound with the corresponding
parent ion m/z are shown on Figs. S3 and S4 (supporting informa-
tion). The retention time for each m/z is the following: adduct 1a:
7 min, adduct 1b: 18 min, adduct 2a: 6.3 min, adduct 2b: 18 min.
In addition, alpha and beta adducts mixtures isolated from the
sep-pak pre-purification were analyzed using optimized condi-
tions. For each mixture of stereoisomers (1a and 1b, 2a and 2b)
the parent ions and their two fragments were detected. These sam-
ples were injected using the LC conditions described above and
monitoring 2 MRM transitions in the MS. The transitions for adduct
1a and 1b were 571.10 > 393.90 and 571.10 > 241.95, and for 2a
and 2b were 528.10 > 393.85 and 528.10 > 243.20 (Figs. S5 and
S6, supporting information). UV spectra of each adduct are shown
in the supporting information section (Figs. S7–S10).
diaminomitosene-DNA adduct using
a
post-oligomerization
method [9,10]. However, our previous efforts to access 1b failed
[9]. Here, we finally report the first chemical synthesis of adduct
1b.
The synthesis of 1b requires the amino precursor 7b (Fig. 2).
Briefly, the amino hydrin precursors were obtained from Mito-
mycin C using HCl (Fig. 2). The 2-amino position was protected
using the trimethylsilylethoxycarbonyl (teoc) group and the
hydroxy group was converted to an azido group in two steps via
a mesylate to give a mixture of stereomers 6a and 6b. At that stage,
both stereomers can be separated by chromatography. Reduction
to the amino group progressed efficiently in the case of the alpha
(trans) amino precursor using triphenylphosphine – Staudinger
reaction [9]. However, the reaction was challenged in the case of
the beta (cis) amino mitosene 7b. Our previous work has shown
that steric hindrance was likely the cause for the sluggish conver-
sion [9]. Indeed, the conversion proceeded rapidly when triphenyl
phosphine was replaced by trimethyl phosphine and the final
amino mitosene 7b was obtained in less than an hour in 93% yield.
Coupling of precursor 7b (cis) with 2-fluoro-O6-(2-p-
nitrophenylethyl)deoxyinosine [18] afforded intermediate 9b and
Fig. 2. Synthesis of 7b.

94
E. Champeil et al. / Bioorganic Chemistry 65 (2016) 90–99
subsequent deprotection at the O6 position using DBU yielded 10b
(Fig. 3). We previously reported that ZnBr2 was a good reagent for
the complete and rapid deprotection of the teoc group [10]. How-
ever, in the case of adduct 9b, these conditions also cleaved the 10-
carbamate group resulting in a mixture of products. Several other
deprotection conditions were tried. Both tetrabutylammonium flu-
oride and tris(dimethylamino)sulfonium difluorotrimethylsilicate
successfully deprotected the teoc group. The pure desired beta
adduct 1b was finally isolated by hexane precipitation followed
by C18 purification (15% acetonitrile in water). UV [11] CD [2b]
and HRMS spectra were recorded (supporting information,
Figs. S13–S15) and its structure was confirmed via co-elution with
an authentic sample prepared by direct alkylation.
Circular dichroism is a published and reliable method to assign
the configuration of mitosene adducts at C1. As can be observed in
the CD spectra of the cis and trans amine (Figs. S32 and S33), com-
pounds 7a and 7b display an opposite cotton effect around 530 nm.
The cis amine (7b, beta) displays a positive CE while the diastere-
omer (7a, alpha) displays a negative CE. The sign is dependent only
of the cis/trans configuration and is not affected by the substituent
at C1 [19]. This effect is also observed for nucleoside–mitosene
adduct pairs [19]. The guanosine chromophore attached at C-1
absorbs at 250–280 nm and the 2 chromophores (mitosene and
guanosine) constitute a coupled oscillator and give rise to coupled
CD in the 200–400 nm. Fortunately, the 530 nm maximum is too
far removed from the purine maxima so that the bands cannot effi-
ciently couple hence this longest wavelength CE is dominated by
the cis/trans (alpha/beta) configuration of the mitosene moiety.
Thus, the presence of a positive CE for adduct 1b around 530 nm
confirms the cis/beta stereochemistry.
This is the first chemical synthesis of a beta mitosene-DNA
adduct. This method paves the way to the beta ICL via the post-
oligomerization method. In this case, the amino precursor 7b will
be coupled to a DNA amonoadduct bearing a fluoroinosine at a
specific site. After annealing, crosslinking under reductive activa-
tion of the mono DNA adduct should afford the desired beta ICL.
3.2. Direct alkylation of deoxyguanosine by MC and DMC
An important feature of the molecular mechanism of action of
the mitomycins is that their original quinone forms are inactive
towards DNA. A reductive activation to the hydroquinone is
required to trigger the monoalkylating and cross-linking activities
of the prodrugs. Spontaneous elimination of methanol generates a
leuco-aziridinomitosene (12) which can follow two paths to gua-
nine N2 alkylation. Path one, an autocatalytic process, generates a
7-aminoaziridine (13) and leads to DNA monoalkylation. Path 2
involves protonation of the leuco-aziridinomitosene (12) to give
a C-1 carbonium ion (14). This intermediate can yield ICLs. The
activated species generated (13 or 14) determine the outcome of
DNA alkylation both with DNA extract and within cells [11].
Fig. 3. Synthesis of 1b.
Table 1
Ratio of monoadducts: alpha versus beta (Ra/b) for direct alkylation reactions. The relative proportion of alpha versus beta deoxyguanosine-N2 monoadducts was calculated using
the ratio of the absorbance at 253 nm of the DNA-adducts (
e253 = 23,900 in 0.01 M potassium phosphate [23a] or e252 = 24,360 in water [23b]). The ratios were determined by
direct HPLC analysis of the reaction mixtures and measurement of the area under the individual isomer peaks. Average values were determined from triplicate experiments.
Solvent
Na2S2O4 (equivalents)
Activation type
MC
DMC
Yield
Yield (%)
Ra/b
Ra/b
Buffer pH 7.4
Buffer pH 5.8
DMSO
DMSO
DMSO
0.27
1.5
0.27
1.5
1
Autocatalytic
Bifunctional
Autocatalytic
Bifunctional
Bifunctional
0.21
–
0.70
0.004
1.65
1.70
–
7.00
Only alpha
6.9
0.58
–
1.32
–
2.01
–
7.77
–
0.16
1.94

E. Champeil et al. / Bioorganic Chemistry 65 (2016) 90–99
95
Fig. 4. Molecular mechanisms of activation of MC and DMC.
Fig. 5. Trans guanosine adduct from bifunctionally activated MC (18a, alpha).
Fig. 6. Cis guanosine adduct from bifunctionally activated MC (18b, beta).
Although all Mitomycin C-DNA adducts obtained upon reductive
activation have been isolated and characterized, questions remain
regarding the activation and the reactivity of both mitomycins [20]
and aziridinomitosenes [21].
When DMC is chemically reduced in the presence of calf thy-
mus DNA or poly(dG.dC)poly(dG–dC), the major adduct obtained
is the alpha (trans) monoadduct, whereas in a cellular environ-
ment, the beta (cis) isomer is produced [4]. Bio-mimetic conditions
favor the autocatalytic pathway – path 1 – because of high concen-
tration of DMC i.e. the major alkylating species is the 7-
aminoaziridine (13). Under intracellular conditions, however, the
alkylating species is believed to be the protonated leucoaziridi-
nomitosene – (14), from path 2 – since low concentrations of drug
disfavor the autocatalytic pathway. Concomitantly, when bifunc-
tional activation – path 2 – is favored in cancer cells treated with
MC – under hypoxic conditions for instance – higher yields of ICLs
and 2,7 DAM adducts – from path 2 – are observed [22].
In order to gain insight into the reactivity of MC and DMC –
independently of the surrounding DNA structure – we generated
either the leuco-aziridinomitosene (14) or the 7-aminoaziridine
(13) in the presence of deoxyguanosine and analyzed the products

96
E. Champeil et al. / Bioorganic Chemistry 65 (2016) 90–99
when DMC is reacted with poly(dG.dC)poly(dG–dC) under autocat-
alytic conditions [4].
In DMSO, autocatalytic reactions required addition of acid to
protonate the aziridine – one equivalent of p-toluenesulfonic acid
was found to be the optimal amount, Table S1, supporting informa-
tion. The overall yield of adducts improved in DMSO versus water
(3.5 times for MC and 2.3 times for DMC) and the ratio Ra/b
increased (MC: Ra/b = 1.70 in water and 7.00 in DMSO; DMC: Ra/
b = 2.01 in water and 7.77 in DMSO). This suggests that the nucle-
ophilic trans attack of either the protonated aziridine (16) or the
open carbocation (17) is enhanced. As was observed in aqueous
conditions, yields of alkylation are higher with DMC than with
MC – 2.7 times higher in buffer and 1.9 times higher in DMSO –
confirming that DMC is a more efficient alkylating agent.
Under bifunctional activation conditions – when 15 is the acti-
vated species – no adduct formation was detected with DMC either
in buffer or DMSO. Other kinetically favored products were formed
(from hydrolysis or reduction). A minute amount of alpha adduct
was detected in DMSO with MC. A plausible explanation for the
extremely low yield is that 15 is so reactive that hydrolysis and/
or reduction products are formed preferentially.
Fig. 7. Trans guanosine adduct from bifunctionally activated DMC (19a, alpha).
Finally, we examined the proportion of adducts generated when
we treated MC and DMC in DMSO with 1 equivalent of sodium
dithionite. Under these conditions, the mechanism of alkylation
is expected to be mainly bifunctional. For MC, we observed a sim-
ilar alpha/beta ratio of products as was found under monofunc-
tional activation conditions (Ra/b = 6.94 versus 7.00). However,
for DMC, the alpha/beta ratio drops dramatically (Ra/b = 1.94 ver-
sus 7.77). This evidences that under bifunctional conditions DMC
has a higher stereopreference for the beta adduct, even in reactions
that do not involve duplex DNA. This may partially explain the for-
mation of beta adducts in a cellular environment. However, with
DMC, the DNA structure seems essential to provide either stabiliza-
tion and/or favorable orientation of the reduced species to generate
the beta adduct as the major product.
3.3. DFT calculations
Next, we investigated the energies of the direct reactions
between deoxyguanosine and the pro-drugs 15, 16 or 17 (Fig. 4)
to appreciate if these different outcomes can be explained by ener-
getics. Energies of the formation of the adducts were calculated by
subtracting the sum of the energies of the reactants from the sum
of the energies of the products – pictures and energies of the differ-
ent entities involved are shown in the supporting information sec-
tion: Figs. S16–S31 and Table S2. After optimization, DFT
calculations established that the product resulting from the alpha
attack of bifunctionally activated MC (15, R = COONH2) with deox-
yguanosine (18a, Fig. 5) is more stable than the beta one (18b,
Fig. 6) and that, in the DMC case, the beta adduct (19b, Fig. 7) is
more stable than the alpha one (19a, Fig. 8) – Table S2, supporting
information.
In addition, during bifunctional activation i.e. when compound
15 is the activated species, formation of the alpha adduct (18a,
Fig. 5) is thermodynamically favored by 2.45 kcal/mol for MC and
formation of the beta adduct (18b, Fig. 8) is favored by 5.08 kcal/-
mol for DMC Table 2. These results correlate with the stereoprefer-
ence exhibited by MC and DMC in a cellular environment, when 15
is believed to be the major alkylating species.
Fig. 8. Cis guanosine adduct from bifunctionally activated DMC (19b, beta).
obtained via HPLC and LC-MS. We reduced MC and DMC using
sodium dithionite for two reasons: (1) reduction is rapid and (2)
it is the reducing agent of choice for the bio-mimetic synthesis of
MC and DMC DNA-adducts. Reactions were run in potassium phos-
phate buffer and in DMSO. Activation type was favored by varying
the amount of sodium dithionite used, hence promoting either the
autocatalytic or the bifunctional pathway.
The relative proportion of alpha and beta adducts (Ra/b) gener-
ated in water or DMSO are presented in Table 1. Under autocat-
alytic conditions – with 16 or 17 as the activated species, Fig. 4 –
reactions in aqueous buffer generate preferentially the alpha
adduct with both MC and DMC. The ratio alpha/beta (Ra/b) is larger
with DMC. This indicates that a trans attack prevails leading to the
formation of the kinetically favored alpha (trans) products rather
than the more stable beta (cis) adducts. The yield of adducts is
higher with DMC; which suggests it’s a more efficient deoxyguano-
sine alkylating agent, as was previously observed [4]. This result
may partially explain why the alpha adduct is the major adduct
Beta attack by deoxyguanosine may be disfavored in the MC
case because of steric hindrance from the carbamate; which results
in the preferential formation of the alpha adduct. With DMC, which
lacks the carbamate at C10, there is no such steric hindrance and
the beta attack is favored. Formation of additional hydrogen bonds
may also favor the beta attack. Alternatively, differences in the
atomic charges as obtained by Mulliken Population analysis may

E. Champeil et al. / Bioorganic Chemistry 65 (2016) 90–99
97
Table 2
Energies of reduction and alkylation reactions.
Reactants
Products
Energy of formation
MC + 2H+ + 2eꢀ
Or
R = OCONH2 (MC case)
E = ꢀ278.19 kcal/mol
R = OH (DMC case)
E = ꢀ267.69 kcal/mol
DMC + 2H+ + 2eꢀ
Reduction
15, R = OCONH2 or R = OH
Trans, alpha
Cis, beta
15 + deoxyguanosine
Bifunctional activation
16 + deoxyguanosine
Autocatalytic activation
17 + deoxyguanosine
Autocatalytic activation
R = OCONH2 (MC case, 18a)
E = ꢀ11.30 kcal/mol
R = OH (DMC case, 19a)
E = ꢀ9.73 kcal/mol
R = OCONH2 (MC case, 18b)
E = ꢀ8.85 kcal/mol
R = OH (DMC case, 19b)
E = ꢀ14.81 kcal/mol
R = OCONH2 (MC case, 1a)
E = ꢀ25.53 kcal/mol
R = OH (DMC case, 2a)
E = ꢀ18.57 kcal/mol
Trans, alpha
R = OCONH2 (MC case, 1b)
E = ꢀ51.82 kcal/mol
R = OH (DMC case, 2b)
E = ꢀ51.29 kcal/mol
Cis, beta
R = OCONH2 (MC case, 1a)
E = ꢀ24.1 kcal/mol
R = OH (DMC case, 2a)
E = ꢀ8.5 kcal/mol
Trans, alpha
R = OCONH2 (MC case, 1b)
E = ꢀ50.3 kcal/mol
R = OH (DMC case, 2b)
E = ꢀ41.2 kcal/mol
Cis, beta
be responsible for the observed stereopreference. The energies of
the reactions of activation for both MC and DMC are quite large
Table 2. This is due to the relieving of the strain present in the azir-
idine which opens upon activation.
The 1a and 1b intermediates as well as the 2a and 2b interme-
diates should not be compared to each other, since they represent
different points on the path. However, it is clear that they feature
higher energies than the final products.
Figures depicting intermediate complexes between MC/DMC
and deoxyguanosine formed during bifunctional activation (path
2) are also shown in Figs. 9–12. Energies of the complexes are
shown in Table 3. These structures represent pseudo-transition
states. The energy of a hydrogen molecule has to be subtracted
from their energy when compared to the final products.
Finally, SN2 type reactions of the protonated aziridine (path 1)
predominantly produces alpha stereochemistry in a kinetic-control
manner as was observed experimentally. From our calculations,
formation of the beta adducts 1b and 2b seems to be favored ther-
modynamically in the case of both MC and DMC under autocat-
alytic conditions when 16 or 17 are the reactive species, Table 2.

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E. Champeil et al. / Bioorganic Chemistry 65 (2016) 90–99
Fig. 9. Pseudo transition state between MC and guanosine toward the formation of
1a.
Fig. 12. Pseudo transition state between DMC and guanosine toward the formation
of 2b.
Table 3
Energies of pseudo transition states between MC/DMC and guanosine.
Pseudo transition state
Energies (ua)
MC-guanosine alpha
MC-guanosine beta
DMC-guanosine alpha
DMC-guanosine beta
ꢀ2026.5257
ꢀ2026.8514
ꢀ1858.1394
ꢀ1858.0312
ent cytotoxicity. We successfully synthesized the first
deoxyguanosine mitosene beta adduct. This is the first time a mito-
sene beta adduct is synthesized using organic chemistry. In the
future, we will study the application of this methodology to the
synthesis of oligonucleotides bearing a cis-adduct. In addition,
we identified and quantified the MC and DMC deoxyguanosine
adducts – trans or alpha versus cis or beta – generated through
direct alkylation to examine the stereopreference exhibited by
the MC and DMC outside the duplex DNA. Yields of deoxyguano-
sine adducts were higher with DMC than with MC – except when
one equivalent of sodium dithionite in DMSO was used – and this
may partially explain why DMC is a more efficient alkylating agent
in cells. Direct alkylation reactions in buffer yielded preferentially
alpha adducts with both MC and DMC. This correlates with previ-
ous observations i.e. only alpha alkylation was observed when
oligonucleotides are reacted with DMC under autocatalytic condi-
tions, contrary to what happens in cells [4]. Bifunctional activation
is believed to be prevalent in a cellular environment and under
these conditions, DMC generates beta adducts preferentially. We
did not observe formation of beta adducts when MC and DMC were
activated bifunctionally and reacted with deoxyguanosine in buf-
fer. This suggests that the duplex DNA structure may stabilize/ori-
ente the activated pro-drugs so that formation of the
thermodynamically favored products is possible [5]. DFT calcula-
tions showed that under bifunctional activation, the thermody-
namically favored adducts are alpha, trans, for MC and beta, cis,
for DMC.
Fig. 10. Pseudo transition state between MC and guanosine toward the formation
of 1b.
Fig. 11. Pseudo transition state between DMC and guanosine toward the formation
of 2a.
However, because the adduct formation is not reversible, the
adducts’ energy cannot be compared to explain the favored
stereochemistry.
Acknowledgments
4. Conclusion
This work was supported by NIH Grant 5SC3GM105460 to Elise
Champeil as well as by the Program for Research Initiatives for
Science Majors (PRISM) at John Jay College. We also want to thank
Dr. Padmanava Pradhan for his help in running CD experiments.
Our lab has been involved in the synthesis of MC and DMC-DNA
adducts in order to understand how/if the isomeric crosslinks gen-
erated by MC and DMC are responsible for the compounds’ differ-

E. Champeil et al. / Bioorganic Chemistry 65 (2016) 90–99
99
[10] E. Champeil, M. Paz, S. Ladwa, C. Clement, A. Zatorski, M. Tomasz, Synthesis of
an oligodeoxyribonucleotide adduct of mitomycin by the
Appendix A. Supplementary material
C
postoligomerization method via a tiamino mitosene, J. Am. Chem. Soc. 130
(2008) 9556–9565.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.02.
003.
[11] G. Suresh Kumar, R. Lipman, J. Cummings, M. Tomasz, Mitomycin C-DNA
adducts generated by DT-diaphorase. Revised mechanism of the enzymatic
reductive activation of mitomycin C, Biochemistry 36 (1997) 1428–14136.
[12] J.A. Bueren-Calabuig, A. Negri, A. Morreale, F. Gago, Rationale for the opposite
stereochemistry of the major monoadducts and interstrand crosslinks formed
by mitomycin C and its decarbamoylated analogue at CpG steps in DNA and
the effect of cytosine modification on reactivity, Org. Biomol. Chem. 10 (2012)
1543–1552.
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