Propargylic sulfone-armed lariat crown ethers: Alkali metal ion- regulated DNA cleavage agents

Article abstract of DOI:10.1006/bioo.1999.1167

Alkali metal ion concentrations within cells are regulated during the cell cycle and may be significantly altered in cancer cells versus normal cells. Thus, the selective destruction of cancer cells might be achieved by agents that marry alkali metal ion

Full text of DOI:10.1006/bioo.1999.1167

Bioorganic Chemistry 8, 98–118 (2000)
doi:10.1006/bioo.1999.1167, available online at http://www.idealibrary.com on
Propargylic Sulfone-Armed Lariat Crown Ethers: Alkali Metal
Ion-Regulated DNA Cleavage Agents
Mark M. McPhee, Jonathan T. Kern, Ben C. Hoster, and Sean M. Kerwin1
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712
Received February 10, 2000
Alkali metal ion concentrations within cells are regulated during the cell cycle and may be
significantly altered in cancer cells versus normal cells. Thus, the selective destruction of
cancer cells might be achieved by agents that marry alkali metal ion recognition elements,
such as crown ethers, with DNA-cleaving moieties. We have prepared a series of propargylic
sulfone-armed lariat crown ethers, which bind sodium and potassium ions and exhibit little
affinity for lithium ions, as determined by a picrate extraction assay. We have investigated
the supercoiled DNA cleavage efficiency of these lariat crown ethers in the presence of var-
ious alkali metal ions. Monomeric propargylic sulfone-armed crown ethers 9b and 10b
cleave DNA at physiologically relevant pH and exhibit modest increases in DNA cleavage
efficiency in the presence of potassium and sodium as compared to lithium. In Tris-containing
buffer, the monomeric lariat crown ether 10b cleaves DNA in a sodium ion-dependent fash-
ion, producing 66% more DNA cleavage in the presence of 5.7 mM sodium than in the
absence of added sodium. The dimeric propargylic sulfone-armed lariat crown ether 11 cleaves
DNA over 10-fold better in the presence of potassium than in the presence of lithium. While
the level of metal ion discrimination exhibited by these compounds is rather modest, they do
represent the first successful attempt to marry molecular recognition of specific alkali metal
ion with covalent modification of DNA.
© 2000 Academic Press
A number of structurally diverse DNA interactive agents form complexes with metal
ions, and metal ion complexation plays an important role in the association of these
molecules with DNA. DNA binding natural products, such as the aureolic acids
mithramycin (1) and chromomycin A3 (2) and the recently isolated cytotoxic bis(ben-
zoxaole) UK-1 (3) bind to DNA as drug-Mg2+ complexes. The quinobenzoxazines, a
class of antitumor fluoroquinolone analogs, form 2:2 drug–Mg2+ dimers in solution, and
this metal ion-induced drug dimerization is required for the DNA binding (4), topoiso-
merase II inhibition (5), and photochemical DNA cleavage (6) exhibited by these com-
pounds. The glycopeptide-derived antitumor antibiotic bleomycins cleave double-
stranded DNA in a process that requires specific metal ions, especially Fe2+ (7). Given
the established role of metal ion recognition in these anticancer compounds, a number
of workers have attempted to design metallo-regulated DNA interactive agents (8).
1 To whom correspondence and reprint requests should be addressed. E-mail: skerwin@mail.utexas.edu.
98
0045-2068/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.

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ALKALI METAL ION-REGULATED DNA CLEAVAGE AGENTS.
Dervan and Griffin have reported the synthesis of a bis-distamycin analog containing
an oligoethyleneglycol linker (9). This DNA affinity cleavage agent displays a unique
DNA sequence cleavage pattern in the presence of Ba2+ or Sr2+, presumably due to the
ability of the DNA-bound host to form a specific metal ion recognition site.
Less well studied are systems in which alkali metal ion recognition is coupled to
DNA binding. Fukuda and coworkers have demonstrated that an anthracene-bearing
15-crown-5 analog displays increased DNA affinity in the presence of K+ or Na+ (10).
Tsukube has proposed the use of lariat crown ethers in the supramolecular assembly
of DNA interactive agents on DNA (11). The polyelectrolyte nature of DNA results
in a high, local concentration of cations in the immediate vicinity of the DNA phos-
phate backbone (12). This locally high concentration of cations may serve to drive the
complexation of weak alkali metal ion ligands attached to DNA interactive moieties.
The effect of complexation can be to increase the affinity of the ligand for DNA or to
induce a reactive conformation of a DNA-cleavage moiety (13).
Alkali metal ion concentrations within cells are regulated during the cell cycle (14),
and may be significantly altered in cancer cells versus normal cells (15–17). This lat-
ter observation may be related to the overexpression (18) or activation (19) of certain
sodium channel proteins within tumor cells. Thus, the selective destruction of cancer
cells might be achieved by agents that marry alkali metal ion recognition elements,
such as crown ethers, with DNA-cleaving moieties.
Nicolaou and coworkers have described propargylic sulfone-containing molecules
as novel DNA-cleaving agents (20). Propargylic sulfones isomerize in mildly basic
medium to allenic sulfones and consequently may serve as reactive electrophiles that
alkylate DNA nucleophiles such as the N-7 of guanine. These alkylated bases may
then undergo Maxam-Gilbert chemistry to effect DNA strand scission (21). Several
groups have investigated the feasibility of modulating the DNA cleavage potential of
propargylic sulfones by controlling the propargylic to allenic isomerization or by the
incorporation of DNA-recognition elements that might increase the affinity of these
molecules for DNA (22).
We previously reported the synthesis and DNA cleavage chemistry of the novel
bis(propargylic) sulfone crown ether 1 (23). Dai and coworkers subsequently report-
ed the synthesis and cytotoxicity of the propargylic sulfone-armed lariat crown ether
2 (24). Crown ether 2 does not display enhanced cytotoxicity relative to noncrown
ether containing analog 3, and the effect of alkali metal ions on the DNA cleavage due
to this crown ether was not reported. More recently, this same group has reported
cleavage due to the propargylic sulfone-armed lariat crown ether 4 (25). Surprisingly,
these authors report no effect of added 1mM Na+, K+, or Sr2+ acetate on the cleavage
of supercoiled φX174 DNA by 4, and an inhibition of DNA cleavage in the presence
of 1 mM Ba(OAc)2.
Herein we report the preparation and metal ion binding properties of propargylic
sulfone-armed lariat crown ethers (9b, 10b) and a model propargylic sulfone (9a,
Scheme 1) that is devoid of alkali metal ion recognition capacity. We also report the
preparation of the potential DNA double strand-cleaving, dimeric bis(propargyl) sul-
fone 11 (Eq. [3]). We additionally report a method for investigating the DNA cleav-
age efficiency of propargylic sulfones in the presence of various alkali metal ions. The
crown ethers 9b, 10b, and 11 are the first compounds reported to possess alkali metal

100
MCPHEE ET AL.
ion-enhanced DNA cleavage activity. The ability of these propargylic sulfone-armed
lariat crown ethers to effect metallo-regulated DNA cleavage contrasts with results
reported by Dai and coworkers, and we offer potential explanations for this disparity.
EXPERIMENTAL SECTION (37)
3-Chloromethylbenzoic acid, ethyl ester (5a) (38). To a solution of 3-chloromethyl-
benzoyl chloride (98% w/w, 0.502 g, 2.6 mmol) in 3 ml of THF was added dropwise via
cannula with stirring a solution of EtOH (470 ml, 8.0 mmol), DMAP (0.066 g, 0.54 mmol),
and pyridine (215 µl, 2.66 mmol) in 2 ml of THF. The reaction mixture was heated under
reflux for 11 h, allowed to cool to room temperature, and transferred to a separatory fun-
nel containing 50 ml of EtOAc and 25 ml of 15:5:5 brine/saturated aqueous NaH2PO4 /
water. The layers were mixed and then separated, and the aqueous layer was extracted with
EtOAc (3 × 15 ml). The combined organic layers were washed with water (15 ml) and
brine (15 ml). The organic layer was dried, concentrated, and the residue was purified by
flash column chromatography on silica gel (20% EtOAc in hexanes) to afford ester 5a
(0.426 g, 83%) as a colorless oil: RF 0.59 (20% EtOAc in hexanes); 1H NMR δ 1.38 (t, J
= 8.6 Hz, 3H), 4.35 (q, J = 8.6 Hz, 2H), 4.59 (s, 2H), 7.4 (t, J = 9.6 Hz, 1H), 7.55 (d, J =
9.6 Hz, 1H), 7.97 (d, J = 9.6 Hz, 1H), 8.03 (s, 1H); 13C NMR d 14.21, 45.44, 61.04, 128.74,
129.4, 129.5, 130.94, 132.79, 137.71, 165.93; IR 1719, 723 cm-1; MS 199 (MH+), 163;
HRMS m/e calcd for C10H12ClO2: 199.0526; found 199.0528.
3-Chloromethylbenzoic acid, 1,4,7,10,13-pentaoxacyclopentadec-2-ylmethyl ester (5b).
To an argon flushed, chilled (-78°C) 10-ml round bottom flask containing a small stir bar, 30,
4 Å molecular sieves and a solution of 2-(hydroxymethyl)-15-crown-5 (95% w/w, 333 mg,
1.26 mmol) and TMEDA (115 µl, 0.762 mmol) in 4.8 ml of CH2Cl2 was added dropwise
with stirring via canula a solution of 3-chloromethylbenzoyl chloride (98% w/w, 252 mg,
1.31 mmol) in 1.2 ml of THF. Stirring was continued for an additional 21.5 h as the reaction
mixture warmed to room temperature. The reaction mixture was transferred to a separatory
funnel containing 50 ml of EtOAc and 40 ml of 30:10 water/saturated aqueous KH2PO4. The
layers were mixed and then separated, and the aqueous layer was extracted with EtOAc (4 ×
50 ml). The combined organic layers were washed with 1:1 water/aqueous saturated KHCO3
(2 × 35 ml). This aqueous wash was extracted with EtOAc (2 × 50 ml) and the pooled EtOAc
layers were dried (K2SO4), concentrated and the residue was purified by flash column chro-
matography on silica gel (10% MeOH in CHCl3) to afford ester 5b (336 mg, 66%) as a pale

ALKALI METAL ION-REGULATED DNA CLEAVAGE AGENTS
101
SCHEME 1
yellow oil: RF 0.56 (10% MeOH in CHCl3); 1H NMR d 3.35–3.62 (m, 16H), 3.62–3.77 (m,
2H), 3.77–3.86 (m, 1H), 4.16 (dd, J = 11.8, 6.5 Hz, 1H), 4.31 (dd, J = 11.8, 4.7 Hz, 1H), 4.44
(s, 2H), 7.26 (t, J = 7.7 Hz, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.88 (s,
13
1H); C NMR d 44.99, 64.58, 69.89, 69.98, 70.06 (3C), 70.23, 70.39 (2C), 70.71, 77.00,
128.39, 129.02, 129.14, 130.19, 132.55, 137.41, 165.25; IR 1730, 714 cm-1; MS 403 (MH+),
367; HRMS m/e calcd for C19H28ClO7: 403.1524; found 403.1519.
4-Chloromethylbenzoic acid, 1,4,7,10,13-pentaoxacyclopentadec-2ylmethyl ester (6b).
To a dry ice-acetone bath-cooled solution of 2-(hydroxymethyl)-15-crown-5 (95% w/w,

102
MCPHEE ET AL.
0.278 g, 1.057 mmol), TMEDA (96 µl, 1.057 mmol), and 4 Å molecular seives (25–30)
in CH2Cl2 (4 ml) was added a solution of of 4-chloromethylbenzoyl chloride (97% w/w,
0.244 g, 1.254 mmol) in 1 ml of CH2Cl2. The resulting solution was allowed to slowly
warm to room temperature over 35 min and stirred at room temperature for an additional
24 h. The reaction mixture was diluted with 35 ml of EtOAc and 30 ml of 5:25 saturated
aqueous KH2PO4 /water, the layers were mixed and then separated, and the aqueous layer
was extracted with EtOAc (4 × 40 ml). The combined organic layers were washed with
1:1 saturated aqueous KHCO3 /water (2 × 30 ml), and these combined aqueous washes
were reextracted with EtOAc (2 × 30 ml). The combined organic layers were dried
(K2SO4), concentrated, and the residue was purified by flash column chromatography on
silica gel (10% MeOH in CHCl3) followed by preparative TLC (10% MeOH in CHCl3) to
afford ester 6b (0.340 g, 80%) as a light pink oil: RF 0.54 (10% MeOH in CHCl3); 1H NMR
d 3.50–3.72 (m, 16H), 3.72–3.85 (m, 2H), 3.85–3.97 (m, 1H), 4.26 (dd, J = 11.8, 6.5 Hz,
1H), 4.41 (dd, J = 11.8, 4.7 Hz, 1H), 4.53 (s, 2H), 7.38 (d, J = 9.4 Hz, 2H), 7.96 (d, J =
9.4 Hz, 2H); 13C NMR d 45.24, 64.87, 70.29, 70.42 (3C), 70.44, 70.58, 70.66, 70.81,
71.01, 77.44, 128.38, 129.95 (2C), 142.21, 165.78; IR 1724, 722 cm-1; MS 403 (MH+),
367; HRMS m/e calcd for C19H28ClO7: 403.1524; found 403.1516.
3-[S-(4-hydroxybut-2-ynyl)thiomethyl]benzoic acid, ethyl ester (7a). A solution of
compound 5a (0.229 g, 1.15 mmol) and thiourea (0.097 g, 1.27 mmol) in 3 ml of EtOH
was heated with stirring under reflux for 21 h. The reaction mixture was then cooled with
an ice-water bath, and n-butylamine (165 µl, 1.67 mmol) was added with stirring. The
reaction mixture was allowed to stir for 2 h as the ice-water bath melted and warmed to
room temperature. The reaction mixture was diluted with 30 ml of CHCl3 and 25 ml of
15:5:5 saturated aqueous KCl/saturated aqueous KH2PO4 /water, the layers were mixed,
separated, and the aqueous layer was extracted with CHCl3 (3 × 10 ml). The organic layer
was dried (K2SO4), concentrated, and the residue was purified by flash column chro-
matography on silica gel (20% EtOAc in hexanes) to afford the thiol (0.166 g, 73%) as a
colorless oil: RF 0.63 (20% EtOAc in hexanes); 1H NMR δ 1.35 (t, J = 8.0 Hz, 3H), 1.76
(t, J = 8.3 Hz, 1H), 3.72 (d, J = 8.3 Hz, 2H), 4.33 (q, J = 8.0 Hz, 2H), 7.34 (t, J = 8.5 Hz,
1H ), 7.47 (d, J = 7.47 Hz, 1H), 7.87 (d, J =8.5 Hz, 1H), 7.95 (s, 1H); 13C NMR δ 14.17,
28.46, 60.86, 128.06, 128.56, 128.90, 130.73, 132.33, 141.34, 166.10; IR 2574, 1721 cm-1;
MS 197 (MH+), 163; HRMS m/e calcd for C10H13O2S: 197.0636; found 197.0633. To a
solution of this thiol (0.097 g, 0.495 mmol) in 3 ml of EtOH was added 4-bromo-2-butyn-
l-ol (38) (99 µl of a 1.13 mg/µl solution in EtOH, 0.95 mmol) with stirring and cooling via
ice-water bath. Diisopropylethylamine (105 µl, 0.092 mmol) was added and, after
5 min, the cooling bath was removed and the reaction mixture was stirred an additional 21
h at room temperature. The reaction mixture was diluted with 25 ml of EtOAc and 25 ml
of 15:5:5 saturated aqueous KCl/saturated aqueous KH2PO4 /water, the layers were mixed,
separated, and the aqueous layer was extracted with EtOAc (3 × 15 ml). The combined
organic layers were washed with water (10 ml) and saturated aqueous KCl (15 ml). The
organic layer was dried (K2SO4), concentrated, and the residue was purified by flash col-
umn chromatography on silica gel (1:1 EtOAc/hexanes) to afford sulfide 7a (0.110 g,
84%, or 61% overall from 5a) as a pale yellow oil: RF 0.55 (1:1 EtOAc/hexanes); 1H NMR
δ 1.38 (t, J = 8.0 Hz, 3H), 2.13 (s(br), 1H), 3.09 (t, J = 2.2 Hz, 2H), 3.88 (s, 2H), 4.30
(t, J = 2.2 Hz, 2H), 4.36 (q, J = 8.0 Hz, 2H), 7.37 (t, J =8.0 Hz, 1H), 7.52 (d, J = 8.0 Hz,
1H), 7.90 (d, J = 8.0 Hz, 1H), 8.03 (s, 1H); 13C NMR d 14.27, 18.91, 35.15, 51.15, 61.14,

ALKALI METAL ION-REGULATED DNA CLEAVAGE AGENTS
103
81.28, 81.90, 128.30, 128.59, 130.29, 130.62, 133.39, 138.00, 166.56; IR 3443, 1716 cm-1;
MS 265 (MH+), 247, 219; HRMS m/e calcd for C14H17O3S: 265.0898; found 265.0896.
3-[S-(4-hydroxybut-2-ynyl)thiomethyl]benzoic acid, 1,4,7,10,13-pentaoxacylopentadec-
2-ylmethyl ester (7b). Following the same general procedure as in the preparation of 7a
above, chloride 5b (0.45 g, 1.12 mmol) gave a residue after workup that was purified by flash
column chromatography on silica gel (10% MeOH in CHCl3) to afford the thiol (0.277 g,
62%) as a light yellow oil: RF 0.60 (10% MeOH in CHCI3); 1H NMR d 1.60–1.94 (s(br), 1H),
3.40–3.62 (m, 16H), 3.64 (s, 2H), 3.65–3.77 (m, 2H), 3.77–3.89 (m, 1H), 4.18 (dd, J = 11.8,
6.5 Hz, 1H), 4.34 (dd, J = 11.8, 4.7 Hz, 1H), 7.25 (t, J = 9.7 Hz, 1H), 7.38 (d, J = 7.6 Hz,
13
1H), 7.76 (d, J = 7.6 Hz, 1H), 7.84 (s, 1H); C NMR d 28.14, 64.54, 69.98, 70.05, 70.15
(2C), 70.32 (2C), 70.44, 70.54, 70.76, 77.13, 127.85, 128.36, 128.76, 130.10, 132.29,
141.16, 165.65; IR 2550, 1724 cm-1; MS 401 (MH+), 367; HRMS m/e calcd for C19H2907S:
401.1634; found 401.1632. This thiol (0.051 g, 0.128 mmol) was alkylated as in the prepa-
ration of 7a above to give a residue after workup that was purified by preparative TLC (1-mm
silica gel plate, 10% MeOH in CHCl3) to afford sulfide 7b (0.049 g, 83%, or 51% overall
from 5b) as a light pink oil: RF 0.50 (10% MeOH in CHCl3); 1H NMR d 2.9 (s(br), 1H), 3.06
(t, J = 2.2 Hz, 2H), 3.51–3.75 (m, 16H), 3.75–3.91 (m, 2H), 3.86 (s, 2H), 3.91–4.02 (m, 1H),
4.28 (dd, J = 11.8, 6.5 Hz, 1H), 4.28 (s(br), 2H), 4.41 (dd, J = 11.8, 4.7 Hz, 1H), 7.37 (t, J =
8.4 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 8.0 (s, 1H); 13C NMR d 18.61,
34.70, 50.63, 64.52, 70.04, 70.11, 70.18, 70.27, 70.34, 70.43, 70.55, 70.71, 70.86, 77.33,
80.38, 82.45, 128.30, 128.60, 129.96, 130.13, 133.49, 138.02, 166.13; IR 3361, 1727 cm-1;
MS 469 (MH+), 403; HRMS m/e calcd for C23H33O8S: 469.1896; found 469.1899.
4-[S-(4-hydroxybut-2-ynyl)thiomethyl]benzoic acid, 1,4,7,10,13-pentaoxacylopentadec
-2-ylmethyl ester (8b). To a 5-ml round bottom flask containing a suspension of thiourea
(28.1 mg, 0.37 mmol) in 400 ml of EtOH was added 4-bromo-2-butyn-1-ol(39) (45 µl of
a 1.13 mg/ml solution in EtOH, 0.343 mmol) with stirring. An argon-flushed reflux con-
denser equipped with a Teflon sleeve over the male joint was attached to the reaction ves-
sel, and the reaction mixture was heated for 14 h at 48°C and then allowed to cool to room
temperature. The reflux condenser was replaced with a septum, and an additional 100 µl
of EtOH was added. The reaction mixture was cooled to 0°C via ice-water bath. n-BuNH2
(34 µl, 0.343 mmol) was added with stirring as the reaction mixture was allowed to warm
to room temperature over 45 min. A solution of chloride 6b (0.138 g, 0.343 mmol) in 400 µl
of EtOH was added slowly via cannula with stirring followed by Hünig’s base (60 µl,
0.343 mmol). The reaction mixture was allowed to stir at room temperature for an addi-
tional 22 h and was then transferred to a separatory funnel containing 70 ml of EtOAc and
62 ml of 42:10:10 saturated aqueous KCl/saturated aqueous KH2PO4 /water. The layers
were mixed, allowed to separate, and the aqueous layer was extracted with 2 × 40 ml of
EtOAc. The combined organic layers were washed with 15 ml of water and 25 ml of sat-
urated aqueous KCl and dried. The residue upon concentration of the organic layer was
purified by preparative TLC (1-mm silica gel plate, 10% MeOH in CHCl3) to afford two
sulfide 8b-containing fractions, A and B: fraction A (lower RF, 75.5 mg, pale yellow oil)
contained pure sulfide 8b; fraction B (36.9 mg) contained 15% w/w sulfide 8b contami-
nated with starting material 6b. The total yield of compound 8b based on recovered 6b was
1
65%. Analytical data for sulfide 8b: RF 0.52 (10% MeOH in CHCl3); H NMR d 2.25
(s(br), 1H), 3.08 (t, J = 2.2 Hz, 2H), 3.52–3.70 (m, 16H), 3.70–3.87 (m, 2H), 3.84 (s, 2H),
3.87–4.0 (m, 1H), 4.27 (dd, J = 11.8, 6.5 Hz, 1H), 4.27 (s(br), 2H), 4.44 (dd, J = 11.8, 4.7

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MCPHEE ET AL.
Hz, 1H), 7.36 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 8.0 Hz, 2H); 13C NMR d 18.85, 35.16, 51.05,
64.77, 70.36, 70.43 (2C), 70.51 (2C), 70.70, 70.79, 70.96, 71.10, 77.58, 81.02, 81.79,
128.79, 128.94, 129.81, 143.00, 166.10; IR 3576, 1725 cm-1; MS 469 (MH+), 451, 401;
HRMS m/e calcd for C23H33O8S: 469.1896; found 469.1904.
General procedure for sulfone formation: 4-[S-4-hydroxybut-2-ynyl)sulfonylmethyl]ben-
zoic acid, 1,4,7,10,13-pentaoxacyclopentadec-2-ylmethyl easter (10b). To an ice-water
bath-cooled solution of sulfide 8b (0.014 g, 0.03 mmol) in 350 µl of MeOH was added drop-
wise with vigorous stirring a solution of Oxone (49.5% w/w KHSO5, 0.13 g, 0.105 mmol) in
350 µl of water and 150 µl of 2.5M, pH 5.5, potassium citrate buffer, and the resulting het-
erogeneous reaction mixture was allowed to stir overnight as the ice-water bath melted. The
reaction mixture was diluted with 25 ml of water and this was extracted with CHCl3 (3 ×
15 ml). The combined organic extracts were washed with water (10 ml) and saturated aque-
ous KCl (15 ml). The residue upon drying (K2SO4) and concentration of the organic layer
afforded sulfone 10b (0.014 g, 91%) as a pale pink oil. Sulfone 10b exhibited some isomer-
ization to the allene after chromatographic purification (silica gel) was attempted via flash
column or preparative TLC. When necessary, compound 10b could be purified via C-18
derivatized silica gel, employing 60:40 MeOH/water as the eluant. Analytical data for sul-
fone 10b: RF 0.40 (10% MeOH in CHCl3); 1H NMR d 3.33 (s (br), 1H), 3.55–3.74 (m, 16H),
3.70 (s(br), 2H), 3.74–3.87 (m, 2H), 3.87–4.0 (m, 1H), 4.32 (dd, J = 11.8, 6.5 Hz, 1H), 4.35
(s(br), 2H), 4.48 (dd, J = 11.8, 4.7 Hz, 1H), 4.49 (s, 2H), 7.55 (d, J = 9.2 Hz, 2H), 8.05 (d,
J = 8.6 Hz, 2H); 13C NMR d 43.84, 50.86, 57.19, 64.89, 70.16 (3C), 70.34 (2C), 70.43, 70.50,
70.75, 70.93, 72.78, 77.36, 87.34, 130.28, 130.97 (2C), 132.43, 165.72; IR 3332,1722, 1329
cm_1; MS 501 (MH+), 433, 369; HRMS m/e calcd for C23H33O10S: 501.1794, found 501.1786.
3-[S-(4-hydroxybut-2-ynyl)sulfonylmethyl]benzoic acid, ethyl ester (9a). Following the
general procedure (see compound 10b), sulfide 7a (0.02 g, 0.076 mmol) gave a residue
after workup that was purified by flash column chromatography on silica gel (1:1 EtOAc/
hexanes) to afford sulfone 9a (0.018 g, 82%) as a colorless, crystalline solid: m.p.
1
79.5–80.5∞C; RF 0.29 (1:1 EtOAc/hexanes); H NMR d 1.39 (t, J = 8.4 Hz, 3H), 2.44
(s(br), 1H), 3.67 (t, J = 2.2 Hz, 2H), 4.36 (t, J = 2.2 Hz, 2H), 4.37 (q, J = 8.4 Hz, 2H), 4.49
(s, 2H), 7.50 (t, J = 8.8 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 8.05 (d, J = 8.8 Hz, 1H), 8.18
(s, 1H); 13C NMR d 14.25, 43.77, 50.97, 57.35, 61.52, 73.08, 87.42, 127.93, 129.30,
130.26, 131.23, 132.25, 135.01, 166.11; IR 3494, 1718, 1316 cm-1; MS 297 (MH+), 279,
251, 163; HRMS m/e calcd for C14H17O5S: 297.0797; found 297.0798.
3-[S-(4-hydroxybut-2-ynyl)sulfonylmethyl]benzoic acid, 1,4,7,10,13-pentaoxapentadec-
2-ylmethyl ester (9b). Following the general method (see compound 10b), compound 7b
(0.048 g, 0.103 mmol) afforded after workup sulfone 9b (0.04 g, 78%) as a colorless oil.
Sulfone 9b exhibited partial isomerization to the allene when chromatographic purification
involving silica gel (preparative silica gel TLC) was attempted. When necessary, compound
9b could be purified via C-18 derivatized silica gel, employing 60:40 MeOH/water as the
1
eluant. Analytical data for sulfone 9b: RF 0.35 (10% MeOH in CHCl3); H NMR d 3.30
(s(br), 1H), 3.53–3.73 (m, 18H), 3.73–3.90 (m, 2H), 3.90–4.04 (m, 1H), 4.33 (dd, J =
11.8, 6.5 Hz, 1H), 4.36 (t, J = 2.2 Hz, 2H), 4.43 (dd, J = 11.8, 4.7 Hz, 1H), 4.48 (s, 2H),
7.48 (t, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 8.17 (s, 1H); 13C
NMR d 43.48, 50.58, 56.85, 64.77, 70.10, 70.15, 70.32, 70.44, 70.62, 70.95, 72.55, 77.31,
88.00, 128.17, 129.33, 130.39, 130.79, 131.89, 135.37, 165.62; IR 3354, 1727, 1331 cm_1;
MS 501 (MH+), 369; HRMS m/e calcd for C23H33O10S: 501.1794; found 501.1802.

ALKALI METAL ION-REGULATED DNA CLEAVAGE AGENTS
105
Compound 11. A stirring solution of sulfide 7b (0.139 g, 0.297 mmol), DMAP (0.018
g, 0.147 mmol) and diisopropylethylamine (48 µl, 0.276 mmol) in 1.1 ml of THF was
cooled with an ice-water bath and malonyl dichloride (97% w/w, 16 µl, 0.16 mmol) was
added dropwise. After 10 min the cooling bath was removed and the reaction mixture
was heated under reflux for 13 h. Upon cooling to room temperature, the reaction mix-
ture was diluted with 75 ml of EtOAc and 50 ml of 30:10:10 saturated aqueous KCl/sat-
urated aqueous KH2PO4/water. The layers were mixed, separated, and the aqueous layer
was extracted with EtOAc (3 × 30 ml). The combined organic layers were washed with
water (20 ml), saturated aqueous KHCO3 (2 × 20 ml), and saturated aqueous KCl (35 ml).
The organic layer was dried (K2SO4), concentrated, and the residue was purified by
preparative TLC (2-mm silica gel plate, 10% MeOH in CHCl3) to give two product-
containing fractions that were each resubjected to purification via preparative TLC (1-mm
silica gel plate, 10% MeOH in EtOAc). The subsequent product-containing fractions
were pooled to afford the corresponding bis(sulfide) (0.029 g, 26% based on recovered
7b (0.035 g)) as a colorless oil: RF 0.52; 0.07 (10% MeOH in CHCl3; 10% MeOH in
EtOAc); 1H NMR δ 3.08 (t, J = 2.2 Hz, 4H), 3.50 (s, 2H), 3.55–3.70 (m, 3H), 3.70–3.87
(m, 4H), 3.85 (s, 4H), 3.87–4.0 (m, 2H), 4.29 (dd, J = 11.8, 6.5 Hz, 2H), 4.44 (dd, J =
11.8, 4.7 Hz, 2H), 4.77 (t, J = 2.2 Hz, 4H), 7.37 (t, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz,
2H), 7.90 (d, J = 8.0 Hz, 2H), 7.97 (s, 2H); 13C NMR δ 18.71, 35.06, 40.89, 53.48, 64.83,
70.34, 70.42, 70.50, 70.53, 70.68 (2C), 70.79, 70.95, 71.09, 76.63, 77.53, 83.10, 128.45,
128.60, 130.11, 130.45, 133.54, 137.91,165.44, 166.10; IR 1770, 1726 cm-1; MS (FAB)
1005 (MH+); HRMS (FAB) m/e calcd for C49H65O18S2: 1005.3612; found 1005.3603.
Following the general procedure for sulfone formation (see Compound 10b; requires 7
equivalents of oxidant to oxidize both sulfur atoms), Oxone oxidation of the bis(sulfide)
(0.017 g, 0.17 mmol) afforded after workup bis(sulfone) 11 (0.016 g, 93%) as a color-
1
less oil: H NMR δ 3.54–3.75 (m, 32H), 3.58 (s, 2H), 3.71 (s(br), 4H), 3.75–3.88 (m,
4H), 3.88–4.02 (m, 2H), 4.32 (dd, J = 11.8, 6.5 Hz, 2H), 4.46 (dd, J = 11.8, 4.7 Hz, 2H),
4.48 (s(br), 4H), 4.83 (s, 4H), 7.48 (t, J = 7.5 Hz, 2H), 7.67 (d, J = 7.5 Hz, 2H), 8.05 (d,
J = 7.5 Hz, 2H), 8.11 (s, 2H); 13C NMR d 40.76, 43.52, 53.08, 57.02, 65.10, 70.31 (3C),
70.48 (2C), 70.61, 70.71, 70.78, 71.07, 75.00, 77.43, 82.08, 127.93, 127.93, 129.28,
130.35, 131.09, 131.98, 135.24, 165.45, 135.59; IR 1770, 1726, 1334 cm-1; MS (FAB)
1069 (MH+); HRMS (FAB) m/e calcd for C49H65O22S2: 1069.3409; found 1069.3396.
Di(4-hydroxybut-2-ynyl)sulfone (12) (18). To a solution of the THP-protected 4-
bromo-2-butyn-1-ol (11) (0.594 g, 2.6 mmol) in 1.7 ml of CH2Cl2 and 0.3 ml of EtOH was
added Na2S Al2O3 (21% w/w Na2S, 0.714 g, 1.9 mmol) in one portion and the reaction
mixture was allowed to stir overnight at room temperature. The reaction mixture was fil-
tered through Celite, the solids were washed with CH2Cl2, and the solvent was evaporat-
ed. The residue was purified by flash column chromatography on silica gel (20% EtOAc
in hexanes) to afford the sulfide (0.182 g, 42%) as a pale yellow oil: RF 0.34 (20% EtOAc
in hexanes); 1H NMR δ 1.42–1.87 (m, 12H), 3.40 (t, J = 2.1 Hz, 4H), 3.46–3.56 (m, 2H),
3.74–3.86 (m, 2H), 4.24 (dt, J = 16.3, 2.1 Hz, 2H), 4.33 (dt, J = 16.3, 2.1 Hz, 2H), 4.76
(t(br), J = 3.7 Hz, 2H); 13C NMR d 18.95, 19.37, 25.24, 30.14, 54.39, 61.88, 79.07, 81.05,
96.69; IR 1120, 1033 cm-1; MS 339 (MH+), 253, 237, 152; HRMS m/e calcd for
C18H27O4S: 339.1630; found 339.1634. To an ice-water bath-cooled solution of sulfide
3.19 (0.055 g, 0.16 mmol) in 4 ml of CH2Cl2 was added m-CPBA (50% w/w, 0.158 g, 0.46
mmol) in one portion and the reaction mixture was allowed to stir at 0°C for 0.5 h and then

106
MCPHEE ET AL.
at room temperature for 5.5 h. The reaction mixture was diluted with EtOAc (50 ml),
washed with saturated aqueous Na2SO3 (2 × 15 ml), saturated aqueous NaHCO3 (2 × 15 ml),
and brine (20 ml) and dried. The solvent was evaporated and the residue was purified by
flash column chromatography on silica gel (1:1 EtOAc in hexanes) to afford sulfone
1
(0.027 g, 45%) as a colorless solid: m.p. 47–49°C; RF 0.57 (1:1 EtOAc/ hexanes); H
NMR δ 1.42–1.87 (m, 12H), 3.46–3.53 (m, 2H), 3.74–3.83 (m, 2H), 4.07 (t, J = 2.1 Hz,
4H), 4.24 (dt, J = 15.9, 2.1 Hz, 2H), 4.33 (dt, J = 15.9, 2.1 Hz, 2H), 4.76 (t(br), 2H); 13C
NMR d 18.96, 25.25, 30.15, 43.63, 54.21, 62.09, 72.78, 84.68, 97.18; IR 1342, 1133, 1035
cm-1; MS 371 (MH+), 285, 269; HRMS m/e calcd for C18H27O6S: 371.1528; found
371.1509. To a solution of this sulfone (0.015 g, 0.04 mmol) in 1.5 ml of EtOH was added
PPTS (0.024 g, 0.096 mmol) and the resulting reaction mixture was heated to 50°C with
stirring for 7 h. Upon cooling to room temperature, the reaction mixture was diluted with
EtOAc (20 ml) and 5:1 brine/water (12 ml). The layers were mixed and separated, and
the aqueous layer was extracted with EtOAc (3 × 10 ml). The combined organic layers
were washed with brine (15 ml) and dried (Na2SO4), and the solvent was evaporated. The
residue was triturated with a small amount of CHCl3 and this was removed to afford sufone
1
12 (5 mg, 63%) as a colorless solid: RF 0.56 (EtOAc); H NMR d 1.65 (s(br), 2H), 4.07
(t, J = 2.0 Hz, 4H), 4.33 (t, J = 2.0 Hz, 4H); 13C NMR (MeOH-d4) δ 44.39, 50.75, 73.04,
87.65; MS 203 (MH+), 185; HRMS m/e calcd for C8H11O4S: 203.0378; found 203.0365.
Alkali metal picrates. Sodium and potassium picrate were prepared according to the
reported method (40). Lithium picrate was prepared by a modification of this method using
lithium hydroxide. The salts were vacuum-dried (300 mm Hg) at 175°C for 2 days and 1H-
NMR analysis (DMSO-d6) indicated that lithium and potassium picrate were anhydrous
while sodium picrate was a monohydrate.
Alkali metal picrate extraction. The general procedure employed was that developed by
Cram (27). Distilled, demineralized water and spectrophotometric grade CHCl3 and
MeCN were used. CHCl3 and water were saturated with each other prior to solution prepa-
rations as a means of preventing volume changes of the phases during the extractions. All
glassware was washed with nonionic detergent, rinsed well with tap water, demineralized
water, and methanol followed by drying in an oven (115°C) or a vacuum dessicator over
P2O5. All operations were conducted at 23–24°C. In a typical extraction, 350 µl of 3.0 mM
aqueous metal picrate and 350 µl of 3.0 mM host in CHCl3 were placed in a 0.5 dram vial
and this was immediately stoppered with a screw-cap. The vials were centrifuged for 1 min
to drive the CHCl3 layer to the bottom of the vial. The vials were then vortexed for 1 min with
a Baxter S/P vortex mixer and centrifuged at high speed for 15 min with an International
Clinical Centrifuge to effect complete phase separation. A 50-µl aliquot (gas tight syringe,
volume measured by difference) of the CHCl3 layer was removed from the middle and bot-
tom of the vial and placed in a 1.0 ml volumetric tube and the volume was brought up to
the mark with MeCN (dilution factor = 20). To ensure no contamination by the aqueous
layer, the syringe needle was washed with a stream of demineralized water and dried with
a Kimwipe before the aliquot was dispensed. The diluted CHCl3 phase aliquot was homog-
enized by several inversions and transferred to a quartz cuvette dedicated for either lithi-
um, sodium, or potassium extractions. Absorbance was measured at 380 nm against a
blank prepared in a manner analogous to the extraction procedure above using demineral-
ized water in place of aqueous metal picrate and host-free CHCl3. The extractability of
metal picrates by CHCl3 in the absence of host was determined as above with CHCl3 that

ALKALI METAL ION-REGULATED DNA CLEAVAGE AGENTS
107
was free of host. The absorbance of the CHCl3 layer (after dilution and against the afore-
mentioned blank) for each metal picrate extracted was found to be 0.002 and this value
was subtracted from all absorbance readings obtained from host-containing extraction
experiments. An aliquot of the host solution in CHCl3 was similarly diluted with MeCN
and its absorbance was measured. This value was also subtracted from the absorbance value
obtained from the extraction experiments.
The concentration of metal picrate in the CHCl3 layer at equilibrium was determined
from the absorbance of the diluted CHCl3 layer with each metal picrate exhibiting an
extinction coefficient (ε) of 16,900 M–1cm–1 at 380 nm in MeCN as determined previous-
ly by Cram and coworkers (41). Complex association constants (Ka) were determined by
the method of Cram (27) from extraction constants (Ke; derived from absorbance meas-
urements made on the CHCl3 layer) and distribution constants (Kd; previously determined
by Cram and coworkers (27); i.e., lithium picrate, 1.42 × 10–3 M–1; sodium picrate, 1.74 ×
10–3 M–1; potassium picrate, 2.55 × 10-3 M–1). The values presented in Fig. 1 represent the
mean plus or minus one standard deviation from two separate extractions.
Distribution of crown ether 9b. Equal volumes (2.53 ml) of a 3mM solution of crown
ether 9b in water-saturated CHCl3 and 3 mM aqueous sodium picrate were homogenized
in a screw-top vial via vortexing for 2 min and the mixture was allowed to stand overnight
to allow complete phase separation. A 2.0-ml aliquot of the upper aqueous layer was care-
fully removed and dried in vacuo; the yellow solid residue was resuspended in CDCl3 and
subsequent 1H NMR analysis revealed no trace of crown ether 9b. Similar 1H NMR analy-
sis of a dried aliquot of the bottom CHCl3 layer indicated the presence of crown ether 9b.
Preparation of alkali metal ion DNA. A strain of DH5α Escherichia coli that contained
the pGAD424 plasmid was incubated in Luria-Bertani media containing 400 µg/ml sodium
ampicillin. Cells were collected and lysed, and the plasmid DNA was isolated with a QIA-
GEN miniprep spin kit according to the manufacturer’s instructions. This afforded the DNA
FIG 1. Alkali metal ion binding by propargylic sulfone-armed lariat crown ethers 9b and 10b and
model compound 9a. Metal ion binding association constants were determined in chloroform using the
picrate extraction procedure. The values for the Li+ ion association for compounds 9a and 9b are upper
limits, as no detectable picrate salt was extracted into chloroform by these compounds.

108
MCPHEE ET AL.
as an aqueous solution in distilled, sterile water. Aliquots (10 µl) of this solution were gen-
tly mixed with 212.2 µl of sterile, pH 7.4, aqueous alkali metal phosphate solution. The
plasmid DNA so obtained was determined spectrophotometrically to be 17 µM base pair
and typically contained 75–85% supercoiled (Form I) DNA. For EC25 experiments, the con-
centration of alkal metal ion before addition of the aqueous DNA solution was 20 mM. For
all other experiments, the concentration of lithium or sodium ions prior to addition of the
aqueous DNA solution was either 0, 1.5, 3.0, or 6.0 mM in 18–20 mM Tris phosphate. It is
worth noting that DNA solutions that were prepared using aqueous alkali metal acetates
exhibited significantly less cleavage upon incubation with propargyic sulfones than solu-
tions prepared as above which employed aqueous alkali metal phosphates.
Quantification of DNA cleavage by propargylic sulfones. In a typical cleavage experi-
ment, 2 µl of a freshly prepared propargylic sulfone solution in d6-DMSO (or, for control
reactions, d6-DMSO that was free of propargylic sulfone) was added to a small, sterile
eppindorf tube followed by 14 µl of an aqueous solution of alkali metal ion-containing
DNA prepared as above. The contents were mixed by brief (5 s) centrifugation at 6000 rpm
and allowed to stand at room temperature for 19 h. Tubes were then heated for 90 s at
70°C. Upon cooling, 2 µl of 8× loading dye (13 mg/ml each of bromophenol blue and
xylene cyanol in 30% aqueous glycerol) was added to each tube, the contents were gently
homogenized, and 5 µl of the resulting solution was loaded onto a 0.7% w/v agarose gel.
The DNA cleavage products were seperated by electrophoresis in 1× TBE running buffer
at 45 V for 2.25 h. The agarose gel was stained for 15 min in TBE buffer that contained
0.25 µg/ml ethidium bromide and then destained in distilled water for 15–30 min. The gel
was scanned with a Molecular Dynamics Fluorimager and the quantities of Forms I, II, and
III DNA were assessed with the ImageQuaNT software program. The degree of cleavage
of Form I DNA was determined using Eq. [1].
2 × Form III + Form II
(
[
]
[
]
Percent cleavage =
× 100
[1]
2 × Form III + Form II + Form I
(
[
]
[
]
[
]
The reported, normalized percentage cleavage accounts for cleavage in control samples
under the reaction conditions employed and this was calculated according to Eq. [2].
( )
% cleavage drug – % cleavage control
(
)
[2]
Normalized percent cleavage =
100 – % cleavage control
The values of normalized percent cleavage of DNA presented in Figs. 3 and 4 represent
the mean plus or minus one standard deviation from three or four separate determinations.
The reported EC25 values were obtained by interpolation from plots of normalized per-
centage cleavage versus concentration. The EC25 values reported represent the average ±
standard deviation for three or more separate determinations.
RESULTS AND DISCUSSION
Synthesis of propargylic sulfone-armed lariat crown ethers 9b, 10b, and 11 and model
propargylic sulfones 9a and 12. The synthesis of the propargylic sulfone-armed lariat
crown ethers began with esterification of the commercially available racemic 2-hydrox-

ALKALI METAL ION-REGULATED DNA CLEAVAGE AGENTS
109
ymethyl-15-crown-5. The ester 5b (Scheme 1) was prepared in 66% yield by the esterifi-
cation procedure of Oriyama and coworkers (26), in which the alcohol is treated with
TMEDA in methylene chloride in the presence of 4 Å molecular sieves at -78C, followed
by addition of 3-chloromethylbenzoyl chloride. The preparation of the para-isomer 6b
proceeded under similar conditions to afford the ester in 80% yield. The model ester 5a
was prepared in 83% yield using standard DMAP-catalyzed esterification procedure.
Transformation of esters 5a, 5b, and 6b to the corresponding benzylic thiols was
accomplished in fair to good yield by nucleophilic displacement by thiourea and sub-
sequent isothiouronium decomposition with n-butylamine (Scheme 1) (27). The thiol
derived from ethyl ester 5a was alkylated with 4-bromobutyn-2-1-ol in the presence
of diisopropylethylamine base to afford the thioether 7a in 61% overall yield from 5a
(Scheme 1). Similarly, chloride 5b was converted to the thioether 7b in 51% overall
yield. Unfortunately, the basic reaction conditions required for isothiouronium salt
decomposition (n-butylamine in EtOH) caused extensive oxidation of the thiol derived
from 6b to the disulfide dimer. This observation led us to attempt a one-pot procedure
in which the benzylic chloride 6b was treated in the presence of diisopropylethy-
lamine with the in situ-formed thiol derived from the treatment of 4-bromo-2-butyn-
1-ol with thiourea followed by n-butylamine. This one-pot, three-step procedure
afforded the thioether 8b in 65% yield.
Oxidation of the propargylic thioethers 7a, 7b, and 8b was accomplished with the
chemoselective reagent Oxone in citrate-buffered aqueous methanol in excellent yield
(Scheme 1). The resulting sulfones 9b and 10b could not be purified by column chro-
matography on silica gel, as isomerization to the inseparable allenic species occurred,
even when acetic acid-containing eluant was employed. Fortunately, the desired propar-
gylic sulfone-armed crown ethers 9b and 10b, as well as the model compound 9a were
obtained essentually pure after aqueous workup, and when necessary, purification could
be accomplished by column chromatography using C-18 derivatized silica gel.
The dimeric crown ether 11 was prepared from the thioether 7b as shown in Eq. [3].
The coupling of 7b to malonyl dichloride was followed by Oxone oxidation to afford 11
in modest overall yield, primarily as a result of incomplete esterification during the cou-
pling step. As in the case of sulfones 9a, 9b, and 10b, dimer 11 was unstable to normal
phase chromatography, but could be purified by reverse phase column chromatography.
[3]
The model bis(propargylic) sulfone 12 was originally reported by Nicalou and cowork-
ers (20). We prepared this compound from the previously reported tetrahydropyranyl
acetal-protected 4-bromo-2-butyn-1-ol (21), which was treated with alumina-supported

110
MCPHEE ET AL.
NaS2 reagent (21), followed by oxidation with mCBPA and deprotection of the alcohol
protecting groups.
Metal ion binding studies of propargylic sulfone-armed crown ethers 9b and 10b and
model propargylic sulfone 9a. Cram’s metal picrate extraction method (28) was used to
assess the alkali metal ion association constants of propargylic sulfone-armed crown ethers
9b and 10b and model propargylic sulfone 9a. The calculated alkali metal ion association
constants for compounds 9a, 9b, and 10b are presented in Fig. 1. As expected, the model
compound 9a was found to be practically devoid of any metal ion binding affinity. In con-
trast, both lariat crown ethers 9b and 10b bind alkali metal ions, with an apparent selec-
tivity of Na+ > K+ >> Li+. This sodium ion selectivity is commensurate with that reported
for picrate-extraction determined binding constants for other 15-crown-5-derived, (substi-
tuted)phenyl-pendent lariat crown ethers (29). Although the picrate extraction technique
for estimating metal ion binding constants is operationally simple and requires minimal
FIGS. 2–5. Agarose gel of DNA cleavage reactions for compounds 9a, 9b, 10b, and 11. Li+ -, Na+-,
or K+ DNA was incubated alone or in the presence of various concentrations of compound for 19 h at room
temperature, briefly heated at 70°C for 90 s, and then subjected to agarose gel electrophoresis. After elec-
trophoresis, the gels were visualized with ethidium bromide staining and UV transillumination and the
image was recorded on a fluorimaging system for quantification. Figure 2: Lanes 1–3, Li+ -, Na+ -, and
K+DNA in the absence of compound; lanes 4–6, Li+DNA in the presence of 0.94, 1.88, and 4.38 mM 9a,
respectively; lanes 7– and 9, Na+DNA in the presence of 0.94, 1.88, and 4.38 mM 9a, respectively; lanes
10–12, K+DNA in the presence of 0.94, 1.88, and 4.38 mM 9a, respectively. Figure 3: Lanes 1–3, Li+-,
Na+-, and K+DNA in the absence of compound; lanes 4–6, Li+DNA in the presence of 1.25, 3.12, and 6.25
mM 9b, respectively; lanes 7–9, Na+DNA in the presence of 1.25, 3.12, and 6.25 mM 9b, respectively;
lanes 10–12, K+DNA in the presence of 1.25, 3.12, and 6.25 mM 9b, respectively. Figure 4: Lanes 1–3,
Li+-, Na+-, and K+DNA in the absence of compound; lanes 4–6, Li+DNA in the presence of 0.31, 0.62, and
0.94 mM 10b, respectively; lanes 7–9, Na+DNA in the presence of 0.31, 0.62, and 0.94 mM 10b, respec-
tively; lanes 10–12; K+DNA in the presence of 0.31, 0.62, and 0.94 mM 10b, respectively. Figure 5: Lanes
1–3, Li+-, Na+-, and K+DNA in the absence of compound; lanes 4–6, Li+DNA in the presence of 0.065,
0.125, and 0.25 mM 11, respectively; lanes 7–9, Na+DNA in the presence of 0.065, 0.125, and 0.25 mM
11, respectively; lanes 10–12, K+DNA in the presence of 0.065, 0.125, and 0.25 mM 11, respectively.

ALKALI METAL ION-REGULATED DNA CLEAVAGE AGENTS
111
FIG. 3.
FIG. 4.
FIG. 5.

112
MCPHEE ET AL.
amounts of material, it suffers from the fact that the value of the binding constants
obtained are a function of the lipophilicity of the crown ether as well as the stability of the
metal ion complex in the organic medium (27). The slight variation in the Ka for sodium
and potassium metal ions, when comparing the positional isomers 9b and 10b is probably
more a reflection of variations in the extraction efficiency due to slight changes in the
lipophilicity of the lariat crown ethers than altered participation of the lariat side chain in
metal ion binding. The aqueous metal ion complex stability constants for these crowns are
no doubt much less than those determined in chloroform using the picrate extraction data.
For 15-crown-5 itself, the aqueous alkali metal ion complex stability constants for Li+,
Na+, and K+ are ~0.0, 5.23, and 8.51, respectively (30).
DNA cleavage studies. To assess the metallo-regulated DNA-cleaving capacity of
these propargylic sulfones, aqueous solutions of supercoiled plasmid DNA in which the
major alkali metal cation species is controlled were prepared. Commercially available
supercoiled phage ΦX174 DNA contains a substantial amount of NaCl. Our repeated
attempts to dialyze these commercially obtained DNA solutions against alkali metal ion
phosphate buffers led to extensive nicking of the supercoiled DNA, presumably due to
mechanical sheering during the dialysis and sample recovery. Therefore, supercoiled
pGAD424 plasmid DNA was isolated from DF5α E. coli using a silica-based column
(QIAGEN Mini-Prep). Elution of the DNA from the column with a minimal volume of
distilled water and dilution of the resulting solution into the appropriate alkali metal ion
phophate buffer (20 mM, pH 7.4) provided supercoiled plasmid DNA solutions in which the
major alkali metal ion in solution is lithium, sodium, or potassium. We refer to these alkali
metal cation-enriched DNA solutions as M+DNAs (i.e., Li+DNA, Na+DNA, or K+DNA).
The propagylic sulfone-armed lariat crown ethers 9b, 10b, and 11, as well as the
model propargylic sulfone 9a and bis(propargylic) sulfone 12 were incubated with
each of these M+DNAs at room temperature overnight (19 h), briefly (90 s) heated to
70°C to enhance the observed cleavage due to DNA alkylation, and then subjected to
agarose gel electrophoresis. The separated DNA forms (supercoiled, relaxed, and lin-
ear) were visualized and quantified by ethidium bromide staining and fluorimaging.
Figures 2–5 show representative DNA cleavage results for compounds 9a, 9b, 10b,
and 11 with Li+DNA, Na+DNA, and K+DNA.
The concentration of compound necessary to effect cleavage of 25% of the super-
coiled DNA (EC25) was determined from plots of percent cleavage vs concentration
for each compound. The EC25s are reported as the mean ± standard deviation for three
or more separate determinations.
The variation in the DNA cleavage EC25s as a function of M+DNA for propargylic
sulfones 9a, 9b, 10b, 11, and 12 are presented in Table 1 and Fig. 6. It is evident from
Fig. 6 that crown ether-containing propargylic sulfones (9b and 10b) exhibit greater
cleavage of Na+DNA and K+DNA than Li+DNA. The propargylic sulfone-armed lari-
at crown ethers 9b and 10b cleave Li+DNA to a similar degree as model propargylic
sulfone 9a, which cleaves all M+DNAs to the same extent, without regard to the dom-
inant cation present. Thus, the cleavage efficiency of propargylic sulfone-armed lari-
at crown ethers 9b and 10b is significantly higher than the model propargylic sulfone
only in the presence of the lariat crown-complexed Na+ and K+ ions. While regioiso-
meric pendent crown ether propargylic sulfones 9b and 10b cleave alkali metal ion-
containing solutions with the same order of efficiency, i.e., Na+DNA K+DNA >>

ALKALI METAL ION-REGULATED DNA CLEAVAGE AGENTS
113
TABLE 1
DNA Cleavage by Propargylic Sulfone-Armed Lariat Crown Ethers
EC25 (mM)a
Compound
Li+DNA
Na+DNA
K+DNA
9a
9b
10b
11
2.2 ± 0.2
2.1 ± 0.2
1.65 ± 0.01
1.1 ± 0.2
2.5 ± 0.3
0.68 ± 0.06
0.45 ± 0.03
0.20 ± 0.01
0.8 ± 0.2
2.3 ± 0.1
0.64 ± 0.09
0.45 ± 0.02
0.09 ± 0.02
1.14 ± 0.05
12
1.29 ± 0.07
a Concentration necessary to effect 25% cleavage of supercoiled DNA, normalized for purity of starting super-
coiled DNA, expressed as an average ± standard deviation for three or more separate determinations.
Li+DNA, the somewhat enhanced potency of 10b versus 9b for all M+DNAs may be
the result of increased binding or steric accessibility of 10b for DNA (22,23).
The exact origin of the specific alkali metal-ion dependent DNA cleavage due to
crown ethers 9b and 10b is not clear. The lack of an effect of the nature of the alkali
metal ion on the DNA cleavage due to model compound 9a argues against a general
alkali metal ion-induced increase in the susceptibility of DNA to alkylation by these
electrophiles. However, we reasoned that the observed alkali metal ion effects could
be a reflection of increased formation of the ultimate allenic sulfone electrophilic
species derived from these crowns in the presence of Na+ and K+ ions. In order to test
this hypothesis, solutions of compounds 9a, 9b, and 10b in 1:1 MeOH/aqueous KHPO4
buffer (100 mM, pH 7.4) were allowed to stand at room temperature for 4 h and the
1
extent of isomerization to the corresponding allenes determined by H NMR. Under
FIG. 6. Effect of alkali metal ions on the supercoiled DNA cleavage efficiency of propargylic sulfone-
armed lariat crown ethers 9b, 10b, and 11 and reference compounds 9a and 12. The EC25 is the micromo-
lar concentration of each compound required to effect 25% cleavage supercoiled DNA when incubated in
Li-, Na-, or K-phosphate buffer, pH 7.4, at 25°C for 19 h.

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MCPHEE ET AL.
these conditions, there was no significant difference in the isomeraztion of the crowns
9b and 10b (50 and 52% isomerization, respectively) as compared to the model
propargylic sulfone 9a (54% isomerization). Thus, the alkali metal ion-facilitated
DNA cleavage by these propargylic sulfone-armed lariat crown ethers must be due to
specific interactions between these compounds and DNA that is enhanced in the pres-
ence of the appropriate metal ion.
The DNA cleavage efficiency of the dimeric crown ether 11 displays a distinct alka-
li metal ion-dependency. As has been reported for other bis(propargylic) sulfones
(18), the dimeric sulfone 11 is a more effective DNA cleavage agent than the propar-
gylic sulfones 9a, 9b, or 10b. Cleavage of Li+DNA by 11 is twice as great as that
observed for the model compound 9a and similar to that observed for the model
bis(propargylic) sulfone 12. The increased DNA cleavage efficiency of dimeric crown
ether 11 for Na+DNA as compared to Li+DNA is slightly greater than that observed
for crown ethers 9b and 10b; however, the relative efficiency of DNA cleavage of 11
in the presence of K+ ions is significantly greater. The K+-dependent DNA cleavage
enhancement observed for 11 is not observed for the simple bis(propargylic) sulfone
12, which shows only a very modest increase in DNA cleavage efficiency for
Na+DNA as compared to either Li+DNA or K+DNA.
Certain 15-crown-5-containing molecules are known to form 2:1 “sandwich” com-
plexes with potassium ions in aqueous solutions (31,32). The linker separating the two
15-crown-5 moieties in compound 11 is sufficiently flexible to allow the formation of
a 1:1 11–K+ complex in which a single K+ ion is sandwiched between the two crown
ether rings (33). The K+ ion-enhanced DNA cleavage due to 11 may reflect the
increased DNA cleavage efficiency of this type of sandwich complex. Alternatively,
the bis(propargylicsulfone) 11 may, in the presence of potassium ions, preferentially
associate with the DNA as a 2:1 11–K+ complex in which a K+ ion is sandwiched by
15-crown-5 moieties from two molecules of 11. Once near the target DNA, the
uncomplexed crown ethers units of the associated complex may recruit additional
molecules of 11 to the DNA via formation of new 2:1 11–K+ sandwich complexes.
The net result may be cooperative binding of 11 to DNA in the presence of potassium
ions and, thus, enhanced cleavage of K+DNA versus Na+DNA or Li+DNA.
Having demonstrated that propargylic sulfone-armed lariat crown ethers exhibit
preferential cleavage of M+DNAs where M+ is Na+ or K+, we next examined whether
the extent of cleavage of supercoiled DNA by these lariat crown ethers could be reg-
ulated by varying the concentration of metal ions in solution. Supercoiled pGAD424
DNA solutions containing 0.0, 1.4, 2.9, or 5.7 mM alkali metal phosphate in Tris
buffer (17–20 mM) were prepared. These DNA solutions were incubated with propar-
gylic sulfones 10b and 9a and the DNA cleavage products were separated and quan-
tified as previously described. The propargylic sulfone-armed lariat crown ether 10b
cleaved supercoiled DNA in a Na+-dependent manner, but the DNA cleavage due to
10b was independent of Li+ ion concentration (Fig. 7). As shown in Table 2, the DNA
cleavage efficiency of lariat crown ether 10b increased 66% on increasing the Na+
concentration in these DNA cleavage reactions from 0 to 5.7 mM. In contrast to these
results, the DNA cleavage due to crown ether 10b was completely unaffected by the
addition of increasing concentrations of Li+ ions (Table 2). Similarly, the DNA cleav-
age due to the model propargylic sulfone 9a, which lacks any metal ion recognition
moiety, is insensitive to increased Na+ ion concentrations in the DNA cleavage reactions

ALKALI METAL ION-REGULATED DNA CLEAVAGE AGENTS
115
FIG. 7. Sodium-ion mediated DNA cleavage due to lariat crown ether 10b. Compound 10b and super-
coiled DNA in TRIS buffer containing Li- or Na-phosphate (20 mM total buffer concentration, pH 7.4)
were incubated for 20 h at 25ºC and the products separated by agarose gel electrophoresis, visualized with
ethidium bromide, and analyzed by quantitative fluorescence imaging. Control lanes are from reactions
containing no added compound, all other lanes are from reactions containing 1.6 mM of lariat crown ether
10b. Significantly more relaxed and linear DNA are formed in the presence of 10b and increasing con-
centrations of Na-phosphate than in the presence of Li-phosphate.
(Table 2). These results conclusively demonstrate that appropriate propargylic sulfone-
armed lariat crown ethers can effect the alkali metal ion-regulated cleavage of DNA.
Dai and coworkers have previously reported the synthesis of the propargylic sul-
fone-armed lariat benzo-15-crown-5 4. These researchers similarly assayed for metal-
lo-regulated cleavage of supercoiled DNA (in this case, ΦX174 phage DNA) by 4 by
the addition of alkali or alkaline earth metal salt solutions (sodium, potassium, bari-
um, or strontium acetate) to solutions containing DNA and 4. When sodium, potassium,
or strontium salt solutions were applied, no change in the extent of DNA cleavage by
4 was observed; the addition of barium salts, however, served to decrease the cleav-
age of DNA by 4. Some commercially available preparations of ΦX174 are supplied
as solutions that contain 12 mM sodium ions. Even after dilution to the working con-
centration for these cleavage assays, the supercoiled DNA may have contained up to
1–2 mM NaCl. In the presence of this Na+-containing DNA solution, Dai’s lariat
TABLE 2
Sodium Ion-Dependent DNA Cleavage by A Propargylic Sulfone-Armed Lariat Crown Ether
Compound
Normalized Percent DNA Cleavage
10b(1.6 mM)
0 mM Li+a
36 ± 2
0 mM Na+a
33 ± 2
1.4 mM Li+a
36 ± 3
1.4 mM Naa
43 ± 3
2.8 mM Li+a
36 ± 1
2.8 mM Na+a
50 ± 3
5.7 mM Li+a
31 ± 1
5.7 mM Na+a
55 ± 3
9a (2.5 mM)
51 ± 2
51 ± 2
51 ± 2
48 ± 5
a Supercoiled DNA in constant ionic strength (20 mM) buffer consisting of TRIS and 0, 1.4, 2.8, or 5.7 mM
Li- or Na-phosphate, pH 7.4.

116
MCPHEE ET AL.
crown may not have responded to further increases in alkali metal ion concentration,
or the changes may have been too subtle to detect without the careful fluorimaging
quantification that we performed. Alternatively, as demonstrated here with dimeric
crown 11 and lariat crowns 9b and 10b, the DNA cleavage efficiency and metallo-
regulation of DNA cleavage exhibited by this class of molecules is somewhat sensi-
tive to structural changes, although the structure activity relationship for this class of
compounds is not yet apparent. Dai’s lariat crown 4 may simply not be an efficient
metallo-regulated DNA cleavage agent, as opposed to compounds 9b, 10b, and 11.
Finally, the inhibition of DNA cleavage due to 4 in the presence of Ba(OAc)2 may be
due to the trapping of the reactive allenic sulfone intermediate by acetate. We have
observed such an effect on the DNA cleavage chemistry of bis(propargylic) sulfone
crown ether 1, in which acetate salts tend to inhibit the DNA cleavage reaction (34).
CONCLUSIONS
Propargylic sulfone-armed lariat crown ethers 9b and 10b, dimer 11 and model
propargylic sulfone 9a were prepared in four to five steps and modest overall yield.
Propargylic sulfone-armed lariat crown ethers 9b and 10b bind sodium and potassium
ions and exhibit little, if any, affinity for lithium ions as determined by a picrate
extraction assay. Model propargylic sulfone 9a did not bind lithium, sodium, or potas-
sium ions to an appreciable extent under the conditions of the picrate extraction assay.
DNA solutions (M+DNAs) have been prepared which contain primarily a single
species of alkali metal ion. Using these DNA solution, we have shown that lariat
crown ethers 9b, 10b, and 11 are effective metallo-regulated DNA cleavage agents.
15-crown-5-containing propargylic sulfones 9b, 10b, and 11 preferentially cleave
Na+DNA and K+DNA versus Li+DNA. The role of specific metal ion recognition in
the metallo-regulated DNA cleavage displayed by 9b, 10b, and 11 is demonstrated by
the equivalent cleavage of Li+DNA by these compounds when compared to the model
propargylic sulfone 9a, which does not recognize metal ions and which displays near-
ly equipotent cleavage of all M+DNA solutions. Most notably, the DNA cleavage due
to lariat crown ether 10b is modulated by the addition of 1–5 mM sodium phosphate
to cleavage reactions containing 20 mM Tris buffer. In comparison, the addition of
lithium phosphate has no effect on DNA cleavage due to 10b.
These propargylic sulfone lariat crown ethers display different metal ion selectiv-
ities in their metallo-regulated DNA cleavage effects. While crowns 9b and 10b
both display over three-fold enhanced DNA cleavage in the presence of either Na+
and K+, when compared to Li+, the bifunctional bis(propargyl sulfone) 11 cleaves
K+DNA over tenfold more efficiently than Li+DNA, and twofold better than Na+DNA.
While this level of metal ion discrimination is not nearly as great as that reported for
alkali metal ion-selective ligands (35), it does represent the first successful attempt
to marry molecular recognition of specific alkali metal ions with covalent modifi-
cation of DNA.
The propargyl sulfone crown ether 11 is a modestly potent DNA cleavage agent. It is
important to note that our DNA cleavage assays are done at a physiologically relevant
pH (7.4) in contrast to the high pH (8.0–8.5) typically employed by others studying
DNA cleavage chemistry of propargylic sulfones (18,19,23,36). Under our more
physiologically relevant conditions, compound 11 is an effective DNA cleavage agent

ALKALI METAL ION-REGULATED DNA CLEAVAGE AGENTS
117
at micromolar concentrations. This compound has been selected by the NCI for fur-
ther evaluation as a potential anticancer agent.
ACKNOWLEDGMENTS
This work was supported by grants from the Petroleum Research Foundation administered through the
American Chemical Society, the Robert A. Welch Foundation, and the US Public Health Service (GM-50892).
Portions of this work have appeared in the Ph.D. dissertation of M. M. McPhee, University of Texas, 1999.
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