Plasmid relaxation induced by copper metalated diglycine conjugates under heterogeneous reaction conditions

Article abstract of DOI:10.1016/S0960-894X(02)01068-5

This paper reports synthesis and plasmid modification activities of a new class of insoluble copper-metalated diglycine conjugates, containing aliphatic linkers of varying length. Besides providing significant rate enhancement for model phosphate ester cl

Full text of DOI:10.1016/S0960-894X(02)01068-5

Bioorganic & MedicinalChemistry Letters 13 (2003) 923–926
Plasmid Relaxation Induced by Copper Metalated Diglycine
Conjugates under Heterogeneous Reaction Conditions
C. Madhavaiah and Sandeep Verma*
Department of Chemistry, Indian Institute of Technology-Kanpur, Kanpur-208016 (UP), India
Received 8 October 2002; accepted 9 December 2002
Abstract—This paper reports synthesis and plasmid modification activities of a new class of insoluble copper-metalated diglycine
conjugates, containing aliphatic linkers of varying length. Besides providing significant rate enhancement for model phosphate ester
cleavage, these constructs also displayed efficient supercoiled plasmid scission, in the absence of co-oxidants, under heterogeneous
catalytic conditions.
# 2003 Elsevier Science Ltd. All rights reserved.
Gly-Gly-His (GGH) tripeptide is a Cu(II) and Ni(II)
binding motif present in serum albumin, neuromedins C
and K, human sperm protamine P2a and histatins,
which aids transport of small molecules and metal ions.1
Metalion coordination abiilty of GGH motif has been
harnessed to engineer semi-synthetic nucleases when
attached to larger protein fragments2 and efforts have
been invested to discover GGH variants based on
metalated diglycine conjugates, by employing variable
length diaminoalkyl linkers, for plasmid modification.
Herein, we present synthesis, characterization and
metalation of four new conjugates 2–5 and their cataly-
tic potentialtowards the celavage of modelP-esters and
a
natural susbtrate, supercoiled plasmid DNA
(pBR322). As these metalloconjugates were insoluble in
common organic solvents and in aqueous buffers, the
present study is a unique example of heterogeneous
catalysis of plasmid nicking reaction.
3
bio-combinatorialapproaches. In another approach,
conjugation of this tripeptide to an array of small
molecules has generated numerous DNA binding and
cleavage reagents.4 Another application of this motif
relates to oxidative protein cross-linking in the presence
of exogenously added co-oxidants, thus making it a
Four diglycine conjugates 2–5 were prepared by reacting
N-benzoylglycylglycine-p-nitrophenylester with diamino-
alkanes such as 1,2-diaminoethane 2, 1,4-diamino
butane 3, 1,8-diamino-3,6-dioxa octane 4 or 1,4-bis-
5
usefulbiochemicaltoo.l
We have truncated the GGH motif to design a diglycine
conjugate (1) and its catalytic assistance towards cleavage
of non-naturalphosphate esters (P-esters) has been
reported.6 This study confirmed that C-terminalhistidine
could be pruned and consequently, a Cu(II) metalated
bis-conjugate (1) effectively catalyzed the cleavage of
modelP-esters: p-nitrophenylphosphate (pNPP), bis-
(p-nitrophenyl)phosphate (bNPP) and 2-hydroxypropyl-
p-nitrophenylphosphate (hNPP), whiel conforming to
the classical Michaelis–Menten kinetic profile. These
results provided us an impetus to further investigate
structure–nucleolytic activity of analogous, copper-
(3-aminopropoxy)butane 5 (Scheme 1)7 and metalated
8
.
with Cu(OAc)2 H2O. The extent of copper incorporation
was determined by atomic absorption spectroscopy and
was reconfirmed by spectrophotometric estimation.9
Amount of copper incorporated was reasonably close
for conjugates 2–5 and values were found to be 272, 278,
282 and 332 mg/g, respectively. These values suggest that
there are approximately 2.5 copper atoms per bis-dipep-
tide ligand. It is not possible to determine the crystal
structures to verify exact stoichiometry and binding
mode due to the insoluble nature of these conjugates.
Pseudo-first-order rate constants (kobs) were determined
for model P-ester cleavage assisted by metalloconjugates
2–5 (Table 1).10 Rate accelerations reaching 106-fold
over the uncatalyzed reaction11 were observed for bNPP.
*Corresponding author. Fax: +91-512-259-7436; e-mail: sverma@
iitk.ac.in
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01068-5

924
C. Madhavaiah, S. Verma / Bioorg. Med. Chem. Lett. 13 (2003) 923–926
Scheme 1. Synthesis of diglycine conjugates 1–5.
Corresponding kobs for a monophosphate ester sub-
strate, pNPP and an RNA model, hNPP, are mentioned
in Table 1. Curiously, all bis-diglycine conjugates
exhibited similar rate enhancements thereby indicating a
lack of linker length effect. This effect could also be
partly ascribed to the fact that all metalated conjugates,
except 5, possess similar copper content. Once again,
the insoluble nature of these constructs play a role in
imparting similar catalytic features to 5, despite the fact
that it contains more Cu2+ than other compounds (2–
4). All unmetalated conjugates were unable to catalyze
the cleavage of model P-esters.
factor was 4.74ꢂ1010 minꢁ1, as determined from the
ꢁln kobs versus 1/T plot (Fig. 1). These results confirm
high stability of these complexes as the catalysts were
able to withstand a temperature of 60 ꢀC, while still
maintaining catalytic assistance for pNPP hydrolysis.
In reaction arrest experiment, conjugate 3 was incu-
bated with bNPP (4 mM) for 13 h and the release of
p-nitrophenolate anion was recorded at regular inter-
vals. After this period, 3 was filtered and change in the
absorbance was further recorded over 75 h. As evident
from Figure 2, cleavage reaction completely aborted
soon after filtration of 3 suggesting a crucialroel for
coordinated copper ions, and lack of the release of
copper ions from the conjugate leading to hydrolysis.
Temperature dependence of the cleavage reactions was
studied by determining the pseudo-first-order rate con-
stants at four different temperatures (Table 2). As
expected, an increase in rate was observed for every
Table 2. Pseudo-first-order constantsa for pNPP hydrolysis at vari-
able temperatures assisted by metalloconjugate 2
10 ꢀC rise in the temperature. The energy of activation
ꢁ1
was found to be 19.75 kcalmol
and the frequency
Temperature (ꢀC)
kobs (minꢁ1
)
30
40
50
60
0.31ꢂ10ꢁ3
0.68ꢂ10ꢁ3
2.77ꢂ10ꢁ3
5.30ꢂ10ꢁ3
Table 1. Pseudo-first-order rate constants for pNPP, bNPP, hNPP
hydrolysis catalyzed by conjugates 2–5a
Substrate
pNPPb
Conjugate
kobs
(minꢁ1
kuncat
(minꢁ1
)
krel
)
aHydrolytic reactions were performed 0.01 M of N-ethylmorpholine
buffer (5 mL) prepared in 50% aqueous methanol, concentration of
pNPP was 7 mM and weight of the conjugate was 1 mg/5 mL, corre-
sponding to 0.87 mM of Cu if the conjugate was to be completely
soluble in buffer.
2
3
4
5
6.79ꢂ10ꢁ4
7.48ꢂ10ꢁ4
5.45ꢂ10ꢁ4
8.08ꢂ10ꢁ4
4.92ꢂ10ꢁ7
7.8ꢂ10ꢁ10
1.98ꢂ10ꢁ6
1.38ꢂ103
1.52ꢂ103
1.11ꢂ103
1.64ꢂ103
bNPPc
hNPPc
2
3
4
5
1.03ꢂ10ꢁ3
0.86ꢂ10ꢁ3
0.80ꢂ10ꢁ3
0.54ꢂ10ꢁ3
1.33ꢂ106
1.11ꢂ106
1.13ꢂ106
0.70ꢂ106
2
3
4
5
1.62ꢂ10ꢁ3
1.60ꢂ10ꢁ3
1.89ꢂ10ꢁ3
1.48ꢂ10ꢁ3
0.81ꢂ103
0.81ꢂ103
0.95ꢂ103
0.75ꢂ103
aHydrolytic reactions were performed in 0.01 M N-ethylmorpholine
buffer prepared in 50% aqueous methanol.
bConcentration of pNPP was 7 mM, weight of conjugates 2–5 were 1 mg/
5 mL, corresponding to 0.87, 0.85, 0.88, 1.04 mM of Cu, if all conjugates
were to be completely soluble in buffer solution (pH 8.0, 40ꢀC).
cConcentrations of bNPP and hNPP were 10 and 3 mM, respectively
and weight of the conjugates 2–5 were 1 mg/3 mL, corresponding to
1.46, 1.43, 1.48, 1.7 mM of Cu, if conjugates were to be completely
soluble in buffer (pH 7.5, 40 ꢀC for bNPP, 30 ꢀC for hNPP).
Figure 1. Plot of ꢁlnKobs versus 1/T, for pNPP hydrolysis catalyzed
by 2.

C. Madhavaiah, S. Verma / Bioorg. Med. Chem. Lett. 13 (2003) 923–926
925
mid relaxation (lanes 6 and 7). These results suggest that
the reaction prefers a pathway that remains uninhibited
by scavenging reagents. However, the reaction was
completely inhibited in the presence of excess EDTA (40
mM) presumably due to copper complexation (lane 8),
reinforcing the importance of coordinated copper ions
in metalated conjugates.
We also performed the cleavage reaction under rigorous
anaerobic conditions13 (argon-filled glove bag) to inves-
tigate the role of ambient oxygen.14 It was found that
the supercoiled plasmid (form I) converted to its closed
circular form (form II) under such conditions (Fig. 6,
lane 2), confirming oxygen-independent cleavage.
Figure 2. Arrest of bNPP hydrolysis catalyzed by conjugate 3.
The results presented here indicate that the plasmid
relaxation is occurring either via non-diffusible radicals
or through hydrolytic pathway. Interestingly, these
constructs oxidatively cleaved plasmid in the presence of
co-oxidants, such as magnesium monoperoxy pthalate
(data not shown). Hydrolytic and oxidative cleavage
duality of copper based artificial nucleases has been
previously discussed.14a,b,15 However, hydrolytic clea-
vage by artificial nucleases is more desirable for nucleic
acid modification as the products so formed are amen-
able to further biochemical manipulations.16
Plasmid modification by copper complexes of tripeptides of
type Xaa-Xaa-His-NH2, have been demonstrated in the
presence of exogenously added co-oxidants such as mag-
nesium monoperoxyphthalate, oxone, and H2O2/ascorbate
system,2b,d presumably through the generation of non-
diffusible reactive species.
We evaluated the reactivity of insoluble metallobis-
dipeptide derivatives using pBR322 supercoiled plasmid
relaxation assay.12 These reactions were performed in
the absence of co-oxidants and thus, the observed
results are likely to have arisen out of non-oxidative
pathway. A time-course analysis of pBR322 cleavage by
conjugate 3 displayed complete conversion of super-
coiled form I to nicked form II, in 8 h (Fig. 3). Analo-
gous to the model P-ester cleavage, a similar profile for
plasmid modification was observed for conjugates 1–5
(Fig. 4), once again suggesting a lack of linker length
effect on the catalytic activities of bis conjugates related
to plasmid relaxation.
Detailed investigations regarding mechanism and clea-
vage product identification are currently under progress.
In short, we have synthesized a series of bio-inspired
nucleolytic reagents that demonstrate efficient plasmid
modification under heterogeneous reaction conditions.
The latter property, imparted to the conjugates by
molecular design, enables their convenient removal at
the end of reaction thus obviating the use of additional
purification steps, prior to subsequent biochemical
transformations. Finally, incorporation of a recognition
element for selective DNA cleavage will also be carried
out and reported in due course of time. It is expected
that such reagents will broaden the scope of chemical
nucleases in many chemico-biological applications.
In order to rule out possible involvement of reactive
species, this reaction was performed in the presence of
radicalscavengers and EDTA ( Fig. 5). Scavengers, such
as t-butanol and DMSO, had no visible effect on plas-
Figure 5. Radical scavenger experiment for pBR322 plasmid cleavage
assisted by 3. Lanes 1–4: pBR322; pBR322 + unmetalated 3; pBR322
+ Cu+2 (50 mM); pBR322 + Cu+2 (50 mM) + EDTA (2 mM),
respectively; lane 5: pBR322 + 3; lanes 6 and 7: pBR322 + 3, in
presence of t-butanol(100 mM) or DMSO (100 mM); al ne 8: pBR322
+ 3, in presence of EDTA (40 mM).
Figure 3. Cleavage of supercoiled pBR322 plasmid DNA by conjugate
3. lane 1: pBR322 (0 h); lanes 2–6: pBR322 + 3 (1, 3, 5, 7, 8 h),
respectively; lane 7: pBR322 (8 h).
Figure 4. Cleavage of supercoiled plasmid pBR322 by metalated con-
jugates. Lane 1: pBR322 (blank); lanes 2–6: pBR322 + conjugates 1–5
(8 h), respectively.
Figure. 6. pBR322cleavage with conjugate 3 under anaerobic condi-
tions. Lane 1: pBR322 + 3 (8 h); lane 2: pBR322 + 3, under anaero-
bic conditions (8 h); lane 3: pBR322 (blank, 8 h).

926
C. Madhavaiah, S. Verma / Bioorg. Med. Chem. Lett. 13 (2003) 923–926
air dried and subjected to atomic absorption and spectro-
photometric analyses to determine copper contents.
Acknowledgements
9. Copper estimation: AAS method: 0.1 g of the sample was
weighed accurately and digested in aqua regia (1 mL) and
heated to 50 ꢀC. Solutions were filtered through Whatman fil-
ter paper (No. 42) and solution was made up to 100 mL using
ultra-pure water. The extract was analyzed using commercially
available standards and a blank solution.
Spectrophotometeric method: The amount of copper incorpo-
rated in conjugates 2–5 was ascertained by digesting a known
amount of sample in 8 M HNO3. A standard curve was pre-
pared by using known concentrations of copper acetate in 8 M
HNO3.
CM thanks Councilfor Scientific and Industrial
Research (CSIR) for award of a SRF. This work was
partially supported by funds from Department of
Science and Technology, India, and the Royal Society
of Chemistry (UK) to one of us (S.V.). An anonymous
referee is also thanked for helpful comments.
References and Notes
1. Harford, C.; Sarkar, B. Acc. Chem. Res. 1997, 30, 123.
2. (a) Mack, D. P.; Iverson, B. L.; Dervan, P. B. J. Am. Chem.
Soc. 1988, 110, 7572. (b) Mack, D. P.; Dervan, P. B. J. Am.
Chem. Soc. 1990, 112, 4604. (c) Mack, D. P.; Dervan, P. B.
Biochemistry 1992, 31, 9399. (d) Harford, C.; Narindrasor-
asak, S.; Sarkar, B. Biochemistry 1996, 35, 4271.
3. (a) Shullenberger, D. F.; Eason, P. D.; Long, E. C. J. Am.
Chem. Soc. 1993, 115, 11038. (b) Muller, J. G.; Hickerson,
R. P.; Perez, J. R.; Burrows, C. J. J. Am. Chem. Soc. 1997,
119, 1501. (c) Qi, Liang.; Ananias, D. C.; Long, E. C. J Am.
Chem. Soc. 1998, 120, 248. (d) Huang, X.; Pieczko, M. E.;
Long, E. C. Biochemistry 1999, 38, 2160.
10. Kinetics: All hydrolytic reactions were performed in
duplicate in centrifuge tubes thermostatted at 40 ꢀC, unless
otherwise mentioned. Totalassay mixture contained 3 or 5 mL
of substrate solution of appropriate concentration prepared in
0.01 M N-ethylmorpholine buffer (pH 8.0 or pH 7.5) in 50%
aqueous solution without metaloconjugate, to correct for
background hydrolysis. Amount of conjugates used for
hydrolysis were 1 mg/5 mL or 1 mg/3 mL of buffer contains
substrate and due to insoluble of these conjugates, copper
concentrations present in 2–5 were taken as catalyst concen-
tration. Initialveolcities were determined from time-depen-
dant release of p-nitrophenolate anion (e=1.65ꢂ104 Mꢁ1
cmꢁ1) and pseudo-first-order rate constants were determined
from (A /A ꢁAt) versus time plots. All reactions were per-
4. (a) Grokhovskii, S. L.; Nikolaev, V. A.; Zubarev, V. E.;
Surovaia, A. N.; Zhuze, A. L.; Chernov, B. K.; Sidorova, N.;
Iu; Zasedatelev, A. S.; Gurskii, G. V. Mol Biol., (Moscow)
1992, 26, 1274. (b) Morier-Teissier, E.; Boitte, N.; Helbecque,
N.; Bernier, J. L.; Pommery, N.; Duvalet, J. L.; Fournier, C.;
Hecquet, B.; Catteau, J. P.; Henichart, J. P. J. Med. Chem.
1993, 36, 2084. (c) Footer, M.; Egholm, M.; Kron, S.; Coull,
J. M.; Matsudaira, P. Biochemistry 1996, 35, 10673. (d) De
Napoli, L.; Messere, A.; Montesarchio, D.; Piccialli, G.; Ben-
edetti, E.; Bucci, E.; Rossi, F. Bioorg. Med. Chem. 1999, 7,
395. (e) Steullet, V.; Dixon, D. W. Bioorg. Med. Chem. Lett.
1999, 9, 2935.
5. (a) Brown, K. C.; Yang, S. H.; Kodadek, T. Biochemistry
1995, 34, 4733. (b) Bertrand, R.; Derancourt, J.; Kassab, R.
Biochemistry 1997, 36, 9703. (c) Brown, K. C.; Yu, Z.; Bur-
lingame, A. S.; Craik, C. S. Biochemistry 1998, 37, 4397.
6. Madhavaiah, C.; Verma, S. Bioconjugate Chem. 2001, 12, 855.
7. Synthesis of conjugates 2–5: N,N0-bis-(N-Benzoylglycylgly-
cine)-1,2-ethanediamine (2). N-benzoylglycylglycine p-nitro-
phenylester (2.24 g, 6.3 mmol) 6 was dissolved in dry DMF (35
mL). A mixture of 1,2-diaminoethane (0.21 mL, 3.5 mmol)
and triethylamine (0.87 mL, 6.3 mmol) in dry DMF (4 mL)
was drop-wise added at room temperature with vigorous stir-
ring, which was continued for 2 h. A precipitate was formed,
which was filtered and washed with MeOH (4ꢂ10 mL) fol-
lowed by acetone (3ꢂ10 mL). The product was recrystallized
from hot DMF (1.35 g, 87%). Similar reaction procedure was
followed to prepare N,N0-bis-(N-benzoylglycylglycine)-1,4-
diamino butane (3), N,N0-bis-(N-benzoylglycylglycine)-1,8-
diamino-3,6-dioxaocatane (4), N,N0-bis-(N-benzoylglycylgly-
cine)-1,4-bis(3-aminopropoxy)butane (5), by using corre-
sponding diaminoalkane linkers.
/
/
formed over eight half-lives.
11. (a) Vance, D. H.; Czarnik, A. W. J. Am. Chem. Soc. 1993,
115, 12165. (b) Breslow, R.; Huang, D. L. Proc. Natl. Acad.
Sci. U.S.A. 1991, 88, 4080.
12. pBR322 plasmid relaxation assay: All cleavage reactions
were performed in sodium cacodylate buffer (10 mM, pH 7.5,
25 ꢀC). Supercoiled plasmid pBR322 (0.2 mg) and metalo-
conjugates (50 mg) were suspended in a totalreaction voul me
of 20 mL. The reactions were terminated by adding the gel
loading buffer (100 mM EDTA, 50% glycerol in Tris–HCl, pH
8.0, 5 mL) and reactions were loaded onto 0.7% agarose gel
containing ethidium bromide (1 mg/mL) and electrophoresed
for 1 h at constant current (80 mA). Gels were imaged on PC-
interfaced Bio-Rad GelDocumentation System 2000.
13. Plasmid cleavage under anaerobic conditions: Nitrogen gas
was purged through cacodylate buffer and it was subjected to
four freeze–thaw cycles. Transfer of reagents and initiation of
cleavage reactions were carefully performed in an argon-filled
glove bag. The reactions were terminated as in ref 12.
14. (a) Ren, R.; Yang, P.; Zheng, W.; Hua, Z. Inorg. Chem.
2000, 39, 5454. (b) Sreedhara, A.; Freed, J. D.; Cowan, J. A. J.
Am. Chem. Soc. 2000, 122, 8814. (c) Borah, S.; Melvin, M. S.;
Lindquist, N.; Manderville, R. A. J. Am. Chem. Soc. 1998, 20,
4557. (d) Hegg, E. L.; Burstyn, J. N. Inorg. Chem. 1996, 35,
7474.
15. (a) Chen, B.; Milne, L.; Landgraf, R.; Perrin, D. M.; Sig-
man, D. S. Chembiochem. 2001, 2, 735. (b) Patwardhan, A.;
Cowan, J. A. Chem. Commun. 2001, 1490. (c) Gilbert, B. C.;
Silvester, S.; Walton, P. H.; Whitwood, A. C. J. Chem. Soc.,
Perkin Trans. 2 1999, 1891. (d) Hettich, R. J.; Roigk, A.;
Schneider, H.-J. Chem. Commun. 1996, 105.
16. (a) Pan, C. Q.; Lazarus, R. A. Biochemistry 1997, 36,
6624. (b) Burke, D. T.; Carle, G. F.; Olson, M. V. Science
1987, 236, 806.
.
8. Conjugates 2–5, were metalated with excess of Cu(OAc)2
H2O in aqueous methanol(25 ꢀC). After 48 h, blue precipitates
so formed were washed with hot DMSO (4ꢂ10 mL), methanol
(4ꢂ10 mL), followed by acetone (4ꢂ10 mL). Conjugates were