A Bis(guanidinium)alcohol Attached to a Hairpin Polyamide: Synthesis, DNA Binding, and Plasmid Cleavage

Article abstract of DOI:10.1002/cbic.201500566

Bis(guanidinium)alcohols have been designed to react with phosphodiester substrates in a fast transphosphorylation step, a quasi-intramolecular process taking place in contact ion pairs. Here the attachment of such compounds to Dervan-type hairpin polyami

Full text of DOI:10.1002/cbic.201500566

DOI: 10.1002/cbic.201500566
Full Papers
A Bis(guanidinium)alcohol Attached to a Hairpin
Polyamide: Synthesis, DNA Binding, and Plasmid Cleavage
´
Daniela Wirth-Hamdoune, Stefan Ullrich, Ute Scheffer, Toni Radanovic, Gerd Dürner, and
Michael W. Gçbel*[a]
Bis(guanidinium)alcohols have been designed to react with
phosphodiester substrates in a fast transphosphorylation step,
a quasi-intramolecular process taking place in contact ion
pairs. Here the attachment of such compounds to Dervan-type
hairpin polyamides is described. The resulting conjugate 1
binds to AT-rich DNA duplexes with affinity similar to that of
the parent polyamide as shown by UV melting experiments
and CD titrations. Conjugate 1 nicks plasmid DNA at concen-
trations ranging from micromolar to high nanomolar.
Introduction
The phosphodiester bonds in DNA are extremely stable against
nucleophilic displacement and exhibit half-lives in the range of
millions of years.[1] Therefore they represent an ideal linker ele-
ment for building up and keeping genetic information. The co-
valent manipulation of DNA, on the other hand, is a big chal-
lenge in which artificial systems can rarely compete with natu-
ral enzymes. Most prominent for genome engineering is the
CRISPR/Cas system, in which Cas9 acts as a programmable
DNA endonuclease, guided by a natural or synthetic RNA
strand.[2] Substrate recognition is in this case achieved through
base pairing.
The most prominent classes of artificial DNA cleavers are
zinc finger nucleases and transcription-activator-like effector nu-
cleases (TALENs).[4] Both use the natural FokI domain as active
catalyst. Entirely synthetic systems are mostly based on metal
ion complexes, in particular those of lanthanides.[3,12] DNA can
also be cleaved by radicals attacking the carbohydrate frame-
work. In this approach redox-active metal complexes such as
EDTA/Fe2+ are used in the presence of oxygen and a reducing
agent.[13]
Our group has previously shown that anion receptor mole-
cules based on bis(guanidinium)alcohols can undergo fast
phosphoryl transfer reactions in the absence of metal ions and
are able to cleave plasmid DNA under physiological condi-
tions.[14] Here we investigate the conjugate 1 (Scheme 1) of
such a compound with a Dervan-type polyamide which was
prepared by liquid-phase synthesis.
The construction of synthetic restriction enzymes generally
requires a sequence recognition element attached to a DNA-
cleaving unit.[3] Specific binding to double-stranded DNA can
be achieved by protein domains,[4] triple-helix-forming oligonu-
cleotides,[5] displacement loops,[6] or pyrrole-imidazole polyam-
ides.[7] The last type of DNA ligand is based on N-methylpyr-
role, N-methylimidazole, and g-aminobutyric acid (GABA). With
the GABA linker the polyamide can fold back as an antiparallel Results and Discussion
hairpin that binds DNA duplexes in the minor grove. With the
Synthesis
aid of different combinations of N-methylpyrrole and N-methyl-
imidazole, specific DNA duplexes rich in AT base pairs can be
recognized. Such compounds are preferentially prepared on
solid support,[7a] although liquid-phase synthesis can be advan-
tageous when larger amounts of product are required.[8] In
recent years polyamides have found their way into a wide
range of applications, such as inhibition of transcription[9] or
sequence-specific alkylation[10] of DNA and also DNA cleav-
age.[11]
Conjugate 1 is based on the bis(guanidinium)alcohol unit (pre-
cursor: 17, Scheme 4, below) and the ImPy-polyamide 16, con-
nected through an amide bond. The two parts were synthe-
sized separately. Compound 17 was prepared as published
previously.[14b] Compound 16 was retrosynthetically divided
into three parts: the two trimers 8b and 10b (Scheme 2) plus
the spacer 14 (Scheme 3).
Polyamide 16 was prepared by liquid-phase synthesis. The
trimers 8b and 10b were assembled first, starting from mono-
mers 2–5.[15,16] Coupling of GABA ester 3 and the activated Py
derivate 2 led to 6 in 91% yield. Hydrogenation at 40 bar (Pd/
C) then reduced the nitro group. The crude product could be
coupled with 2, and O2N-PyPy-GABA-OMe (7) precipitated
from the reaction mixture (80%). Compound 7 was hydrogen-
ated again and formed trimer 8a on treatment with imidazole
5 (83%). Ester hydrolysis finally led to 8b (93%).
´
[a] D. Wirth-Hamdoune, Dr. S. Ullrich, Dr. U. Scheffer, T. Radanovic,
Dr. G. Dürner, Prof. Dr. M. W. Gçbel
Institut für Organische Chemie und Chemische Biologie
Johann Wolfgang Goethe-Universität Frankfurt
Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany)
E-mail: M.Goebel@chemie.uni-frankfurt.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201500566
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Scheme 1. Structure of the DNA cleaving conjugate 1 (counter ion: chloride).
Scheme 2. a) DIEA, CH2Cl2, 2, 3, 16 h, RT, 91%; b) i. Pd/C, MeOH, 2 h, 40 bar H2, 558C; ii. NaH, DIEA, CH2Cl2, 2, 16 h, RT, 80%; c) i. Pd/C, MeOH, 3 h, 40 bar H2,
608C; ii. DIEA, CH2Cl2, 5, 16 h, RT, 83%; d) LiOH·H2O, MeOH/H2O, 16 h, 08C!RT, 93%; e) i. 4, Pd/C, EtOAc, 3 h, 40 bar H2, 558C; ii. DIEA, CH2Cl2, 2, 16 h, RT,
85%; f) i. Pd/C, EtOAc, 3 h, 40 bar H2, 558C; ii. DIEA, CH2Cl2, 2, 16 h, RT, 75%; g) TiCl4, CH2Cl2, 90 min, 08C!RT, 81%.
In a similar way monomer 4 was reduced to the correspond-
ing amine and acylated with compound 2. O2N-PyPy-OtBu 9
precipitated and was isolated by filtration (85%). Further re-
duction and acylation with 2 converted 9 into trimer 10a
(75%). Ester hydrolysis was achieved with TiCl4 and yielded
acid 10b (81%).
79% yield (based on Boc2O) after distillation. The residual
amino group of 12 was then allowed to react with Cbz-b-Ala-
OH to give 13 (87%). Partial deprotection with HCl followed
by condensation with Boc-GABA produced linker 14 (87%).
Assembly of the three building blocks (Scheme 4) began
with removal of Cbz from linker 14 and coupling with
acid 10b. The small excess of 10b was removed by column
chromatography to allow the isolation of 15 in 72% yield.
The starting material for the synthesis of 14 was compound
11, which was first treated with Boc anhydride to afford 12 in
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formation) in spite of the presence of two extra guanidinium
ions.
DNA cleavage
For all cleavage studies the supercoiled plasmid pUC19 was
used in 50 mm HEPES·NaOH buffer and analyzed by agarose
gel electrophoresis. This technique separates and quantifies
the supercoiled (form I), the nicked (form II), and the ring-
opened form of the plasmid. pUC19 consists of 5372 nucleo-
tides. Cleavage of any of the phosphodiester bonds and frag-
mentation of any of the nucleotides will convert supercoiled
DNA into nicked DNA. The assay thus integrates thousands of
different reaction channels. This fact and the strain of the su-
percoiled structure make plasmids quite sensitive to hydroly-
sis—in contrast to the exceptional stability of individual phos-
phodiester bonds.
Scheme 3. a) Boc2O, 1,4-dioxane, RT, overnight, 79%; b) Cbz-b-Ala, DIEA,
HOBt, DIC, DMF, 608C, 4 h, 87%; c) i. AcCl, MeOH, quant.; ii. Boc-GABA, DIEA,
HOBt, DIC, DMF, 808C, 4 h, 87%.
Then, the nitro group of 15 was reduced, and condensation
with 8b completed the synthesis of the hairpin amide (43%,
higher yields are accessible in this step). Removal of Boc and
condensation with acid 17 produced compound 18 (62%).
After a final deprotection step, treatment with 4,5-dihydroimi-
dazole-2-sulfonic acid[17] led to the target compound 1 as a yel-
lowish foam after HPLC purification (19%).
Nonconjugated bis(guanidinium)alcohols such as 20 must
be present in high-millimolar concentrations to nick pUC19 ef-
fectively.[14b] In the first set of experiments the conversion of
supercoiled DNA into nicked DNA was determined as a function
of cleaver concentration (Figure 1): 30–40% conversion was
Binding to DNA duplexes
Hairpin polyamides such as 19 are known to bind double-
stranded DNA rich in AT base pairs in the minor groove and to
effect considerable duplex stabilization. The duplex d(CATTGT-
TAGAC)3’:d(GTCTAACAATG)3’, for example, has a reported Tm
value of (42.7Æ0.3)8C that rises to (53.7Æ0.7)8C upon addition
of one equivalent of polyamide 19.[18] To see if the presence of
the bis(guanidinium) unit might alter the DNA affinity we re-
peated the melting experiment under identical conditions (see
the Experimental Section and the Supporting Information) in
the absence (42.0Æ0.2)8C and in the presence (53.6Æ1.5)8C
of conjugate 1. The conformity of the results shows that the
two compounds 1 and 19 must have similar DNA affinities.
Hairpin polyamides 1, 16, and 19 are all achiral and there-
fore do not show any Cotton effect. However, upon binding to
DNA the chiral environment is expected to induce CD bands in
the region around 330 nm in which the UV absorption of the
polyamides is not overlain by the absorption bands of DNA.
Such induced CD bands are sensitive to local conformations.
CD spectra of conjugate 1 bound to d(CATTGTTA-
GAC)3’:d(GTCTAACAATG)3’ (Supporting Information) look very
similar to analogous spectra reported for compound 19.[18] The
bis(guanidinium) alcohol present in 1 apparently does not dis-
turb the structure of the polyamide·DNA complex. When DNA
is titrated with polyamides 1 or 19, the intensities of induced
CD bands as a function of ligand concentration also give
access to binding constants. A value of (7.3Æ1.3)”106 mÀ1 has
been reported for 19 and the 11-mer DNA duplex mentioned
above.[18] Analogous titrations with conjugate 1 show a slightly
reduced value of Ka ((1.5Æ0.6)”106 mÀ1, see the Supporting In-
Figure 1. Concentration dependence of pUC19 DNA cleavage by 1. Condi-
tions: 22 nm DNA, 50 mm HEPES·NaOH (pH 7.0), 378C, 20 h; electrophoresis
on 1% agarose gel, ethidium staining; inverted grayscale. I: plasmid-DNA
form I. II: nicked circular DNA form II. Lane 1: unincubated control. Lanes
2–16: 0, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.02, 0.01, 0.005,
0.002, and 0.001 mm, respectively (see the Supporting Information for a full-
size picture of the gel).
achieved by conjugate 1 at concentrations 2000 times lower
than those required for the unconjugated analogue 20 to give
comparable effects. No linear DNA was observed under such
conditions. Cleavage effects above background are seen at
conjugate concentrations as low as 78 nm, at which no more
than three or four molecules of compound 1 are present per
copy of plasmid. Plasmid cleavage by unconjugated bis(guani-
dinium)alcohols becomes more effective with increasing pH
values.[14b] In contrast, conjugate 1, when tested between pH 7
and 9, works best in more acidic buffers (Figure 2).
Basic conditions could deprotonate the charged amino
group of 1. However, only a minor reduction in DNA affinity is
observed upon removal of the terminal amine from compound
19.[19] Thus, linker deprotonation cannot fully explain the
change in reactivity.
Finally, the time course of plasmid cleavage was determined
(Figure 3). After 8 h, conjugate 1 had converted nearly 30% of
the plasmid into the nicked form, not much less than the 40%
obtained after 20 h. The reason why the reaction stalls or goes
into saturation is presently not known.
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Scheme 4. a) i. 14, Pd/C, MeOH, 40 bar H2, 558C, 3 h; ii. 10b, DIEA, HOBt, DIC, DMF, 608C, 4 h, 72%; b) i. Pd/C, MeOH, 40 bar H2, 508C, 2 h; ii. 8b, DIEA, HOBt,
DIC, DMF, 608C, 3 h; iii. AcCl, MeOH, 43%; c) 17, DIEA, HOBt, DIC, DMF, 608C, 3 h, 62%; d) i. AcCl, MeOH; ii. 2:1 MeOH/NEt3, 4,5-dihydroimidazole-2-sulfonic
acid, RT, 16 h, 19%.
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Information). We cannot rule out the possibility that such inter-
mediates might undergo a fast religation step, thus making
isolation of covalent intermediates a very difficult task. DNA
cleavage induced by naphthoxy radicals is another mechanistic
option. It is not excluded but seems less probable because
DNA cleavage by 20 has been shown not to depend on oxy-
gen.[14b] Finally, the hydroxy groups of 1 and 20 might act as
general acids to destroy DNA by depurination. Further experi-
ments to localize the cleavage sites of 1 are underway. A
purine-specific cleavage pattern, for example, would point to
an acidolytic mechanism whereas phosphoryl transfer would
be expected to form covalent intermediates and not to show
a preference for purines. The observation that 20 cleaves
pUC19 more rapidly at higher pH, however, argues against
acidolytic depurination. Thus, at present a transphosphorylation
mechanism looks probable for conjugate 1. However, further
studies are required to see whether 1 cleaves DNA in a reversi-
ble way or even behaves as an artificial DNA topoisomerase.
Figure 2. Plasmid cleavage as a function of pH: 22 nm pUC19 DNA, 10 mm
conjugate 1, 50 mm HEPES·NaOH, pH as given above, 378C, 20 h. Plasmid
DNA is unstable in HEPES buffers below pH 7 (Figure S7). Mean values of
three experiments. (Gel is shown in the Supporting Information.)
Experimental Section
General: Flash column chromatography was performed with silica
gel 60 (0.032–0.063 mm) purchased from Macherey–Nagel or Roth.
Thin-layer chromatography (TLC) was performed with Merck 60
F254 silica gel plates (0.2 mm layer) with visualization under UV radi-
ation (254 nm) or by staining with an acidic aqueous ammonium
molybdate(IV) or ninhydrin solution; 1H NMR (300 K): Bruker
AM 250 operating at 250 MHz or Bruker AM 300 operating at
300 MHz. Chemical shifts in ppm. The solvent peaks were calibrat-
ed to 7.25 ppm (CDCl3) or 2.50 ppm ([D6]DMSO). 13C NMR (com-
plete proton decoupling): Bruker AM 250 operating at 60 MHz or
Bruker AM 300 operating at 75 MHz. The solvent peaks were cali-
brated to 77.2 ppm (CDCl3) or 39.4 ppm ([D6]DMSO). COSY, HSQC,
HMBC, and DEPT experiments were used to support the NMR as-
signment. Mass spectra were recorded with a Fisons VG Platform II
(ESI) or a Fisons VG TOFSpec (MALDI). FTIR: PerkinElmer Spectrum
Two. Melting points are not corrected. HPLC: Pump Waters 590
and detector Waters 440. DIC=N,N-diisopropylcarbodiimide,
DIEA=diisopropylethylamine, Hex=n-hexane, HOBt=1-hydroxy-
benzotriazole (>86%).
Figure 3. Plasmid cleavage as a function of time: 22 nm pUC19 DNA, 10 mm
conjugate 1, 50 mm HEPES·NaOH pH 7.0, 378C, reaction time as given
above. Mean values of three experiments. (Gel is shown in the Supporting
Information.)
Conclusions
Bis(guanidinium)alcohols have been designed to bind anionic
phosphodiesters as contact ion pairs and to undergo fast
phosphoryl transfer in which their hydroxy groups act as nu-
cleophiles. In reactions with activated model substrates the
resulting phosphorylation products can be isolated and fully
characterized.[14] The naphthol OH group present in com-
pounds 1 and 20 is also a good leaving group, which makes
the phosphorylation step potentially reversible. In a formal
sense bis(guanidinium)alcohols can be seen as models of
type I DNA topoisomerases, enzymes that reversibly cleave and
ligate DNA strands without consumption of ATP. They rely on
tyrosine residues as nucleophiles and leaving groups in combi-
nation with arginine residues used for substrate binding and
activation.[20] Reversible phosphorylation of tyrosine is also the
mechanism of many recombinases and integrases.[21] In con-
trast to compound 20, which requires millimolar concentra-
tions for DNA cleavage, the polyamide conjugate 1 works
down to the nanomolar range. In analogy with the reactions
between 20 and model substrates (e.g., bis-(4-nitrophenyl)-
phosphate), the products formed by 1 and DNA might be
mixed phosphodiesters of its naphthol hydroxy group with oli-
gonucleotide 5’- or 3’-phosphates. However, the mechanism is
not yet confirmed (for unsuccessful studies, see the Supporting
Synthesis
O2N-Py-GABA-OMe (6): DIEA (6.69 mL, 39.06 mmol) was added to a
suspension of methyl g-aminobutyrate·HCl (3,[16] 4.00 g,
26.04 mmol) in CH2Cl2 (35 mL). Subsequently, 2[22] (7.78 g,
28.64 mmol) was added. After the system had been stirred for 16 h
at room temperature, the solvent was evaporated under reduced
pressure, and the crude product was purified by column chroma-
tography (Hex/EtOAc 1:2) to afford 6 as a colorless solid (6.36 g,
91%). M.p. 1208C; 1H NMR (250 MHz, [D6]DMSO): d=8.39 (t, J=
5.7 Hz, 1H; NH), 8.11 (d, J=2.0 Hz, 1H; Py-H), 7.42 (d, J=2.1 Hz,
1H; Py-H) 3.90 (s, 3H; NÀCH3), 3.58 (s, 3H; COOCH3), 3.21 (q, J=
6.8 Hz, 2H; CH2), 2.36 (t, J=7.4 Hz, 2H; CH2), 1.75 ppm (quin, J=
7.1 Hz, 2H; CH2); 13C NMR (60 MHz, [D6]DMSO): d=173.0, 159.8,
133.7, 127.7, 126.4, 107.2, 51.2, 37.9, 37.2, 30.7, 24.3 ppm; IR: n˜ =
3391, 3131, 2945, 2885, 1731, 1662, 1543, 1520, 1506, 1485, 1465,
1450, 1427, 1408, 1353, 1312, 1280, 1197, 1172, 1132, 1100, 1053,
979, 893, 849, 810, 745, 708, 587, 521, 504, 458 cmÀ1; MS (ESI+): m/
z: calcd for C11H15N3O5 +H+: 270.1 [M+H]+; found: 270.5; elemen-
tal analysis calcd (%) for C11H15N3O5: C 49.07, H 5.62, N 15.61;
found: C 48.96, H 5.40, N 15.63.
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O2N-PyPy-GABA-OMe (7): Compound 6 (2.00 g, 7.40 mmol) was
added to a suspension of Pd/C (0.30 g, 10% on active charcoal) in
anhydrous MeOH (30 mL). The mixture was stirred in a steel auto-
clave under H2 (40 bar) for 2 h at 558C. The catalyst was removed
by filtration through Celite, and the solvent was evaporated under
reduced pressure. After drying of the product in vacuo, the prod-
uct was dissolved in CH2Cl2 (10 mL). The resulting solution was
dropped into a solution of 2 (2.40 g, 8.80 mmol) in CH2Cl2 (30 mL).
After that NaH (60% in mineral oil, 0.29 g, 7.40 mmol) was added
to the solution in portions over 30 min, followed by DIEA (1.91 mL,
11.14 mmol). During stirring for 16 h at room temperature a yellow
solid precipitated. Water (20 mL) was carefully added to the sus-
pension. The suspension was filtered, and the residue was washed
with H2O, CH2Cl2, and MeOH to yield 7 as a yellow solid (2.31 g,
temperature. The solvent was evaporated, and the crude product
was purified by column chromatography (CH2Cl2/MeOH 5:1) to
yield 8b as a colorless solid (0.90 g, 93%). M.p. 1748C; 1H NMR
(250 MHz, [D6]DMSO): d=12.02 (brs, 1H; COOH), 10.41 (s, 1H; NH),
9.89 (s, 1H; NH), 8.02 (t, J=5.5 Hz, 1H; NH), 7.38 (s, 1H; Im-H), 7.28
(d, J=1.6 Hz, 1H; Py-H), 7.18 (d, J=1.7 Hz, 1H; Py-H), 7.16 (d, J=
1.7 Hz, 1H; Py-H), 7.04 (s, 1H; Im-H), 6.88 (d, J=1.7 Hz, 1H; Py-H),
4.00 (s, 3H; NÀCH3), 3.85 (s, 3H; NÀCH3), 3.80 (s, 3H; NÀCH3), 3.19
(q, J=6.5 Hz, 2H; CH2), 2.25 (t, J=7.5 Hz, 2H; CH2), 1.71 ppm
(quint., J=7.1 Hz, 2H; CH2); 13C NMR (60 MHz, [D6]DMSO): d=
174.5, 161.3, 158.5, 156.1, 138.8, 127.0, 126.3, 123.1, 123.0, 122.1,
121.4, 118.6, 117.8, 104.9, 104.3, 37.9, 36.1, 35.9, 35.1, 31.4,
24.8 ppm; IR: n˜ =3377, 3302, 2951, 1733, 1714, 1624, 1582, 1540,
1520, 1469, 1428, 1400, 1357, 1269, 1206, 1172, 1123, 1063, 1006,
935, 886, 821, 797, 776, 762, 696, 673, 635, 614 cmÀ1; MS (ESI+):
m/z: calcd for C21H25N7O5 +H+: 456.2; found: 456.3 [M+H]+, 478.4
[M+Na]+.
1
80%). M.p. 2008C; H NMR (250 MHz, [D6]DMSO): d=10.24 (s, 1H;
NH), 8.19 (d, J=1.6 Hz, 1H; Py-H), 8.09 (t, J=6.7 Hz, 1H; NH), 7.59
(d, J=2.0 Hz, 1H; Py-H), 7.22 (d, J=1.9 Hz, 1H; Py-H), 6.87 (d, J=
1.9 Hz, 1H; Py-H), 3.97 (s, 3H; NÀCH3), 3.82 (s, 3H; NÀCH3), 3.60 (s,
3H; COOCH3), 3.20 (q, J=6.7 Hz, 2H; NCH2), 2.36 (t, J=7.4 Hz, 2H;
CH2), 1.75 ppm (quin, J=7.0 Hz, 2H; CH2CH2CH2); 13C NMR (60 MHz,
[D6]DMSO): d=173.1, 161.2, 156.9, 133.8, 128.1, 126.3, 123.2, 121.3,
118.0, 107.5, 104.1, 51.2, 37.7, 37.4, 35.9, 30.8, 24.6 ppm; IR: n˜ =
3380, 3333, 3097, 2924, 2854, 1716, 1671, 1644, 1562, 1531, 1492,
1467, 1438, 1415, 1402, 1380, 1360, 1334, 1300, 1250, 1209, 1166,
1144, 1114, 1082, 1059, 1008, 985, 964, 890, 867, 807, 777, 757, 748,
717, 681, 660, 621, 590, 582, 490, 462 cmÀ1; MS (ESI+): m/z: calcd
for C17H21N5O6 +H+: 392.2; found: 392.2 [M+H]+, 414.2 [M+Na]+;
HRMS (MALDI) m/z: calcd for C17H21N5O6: 391.14863 [M]; found:
391.1487.
O2N-PyPy-OtBu (9): Compound 4[15] (4.00 g, 17.68 mmol) was added
to a suspension of Pd/C (0.40 g, 10% on active charcoal) in anhy-
drous EtOAc (30 mL). The mixture was stirred in a steel autoclave
under H2 (40 bar) for 3 h at 558C. The catalyst was removed by fil-
tration through Celite, and the solvent was evaporated. After
drying of the product in vacuo, the product was dissolved in
CH2Cl2 (10 mL). The resulting solution was added dropwise to a
solution of 2 (5.28 g, 19.45 mmol) in CH2Cl2 (30 mL), followed by
DIEA (4.54 mL, 26.52 mmol). During stirring for 16 h at room tem-
perature a yellow solid precipitated. The suspension was filtered,
and the residue was washed with CH2Cl2 to yield 9 as a yellow
1
solid (5.23 g, 85%). M.p. 217–2188C; H NMR (250 MHz, [D6]DMSO):
ImPyPy-GABA-OH (8b): Compound 7 (2.00 g, 5.11 mmol) was added
to a suspension of Pd/C (0.30 g, 10% on active charcoal) in anhy-
drous MeOH (30 mL). The mixture was stirred in a steel autoclave
under H2 (40 bar) for 3 h at 608C. The catalyst was removed by
filtration through Celite, and the solvent was evaporated. After
drying of the product in vacuo, the product was dissolved in
CH2Cl2 (10 mL). The resulting solution was dropped into a solution
d=10.20 (s, 1H; NH), 8.17 (d, J=1.9 Hz, 1H; Py-H), 7.56 (d, J=
2.1 Hz, 1H; Py-H), 7.38 (d, J=1.9 Hz, 1H; Py-H), 6.83 (d, J=2.0 Hz,
1H; Py-H), 3.95 (s, 3H; NÀCH3), 3.81 (s, 3H; NÀCH3), 1.50 ppm (s,
9H; C(CH3)3); 13C NMR (60 MHz, [D6]DMSO): d=159.8, 156.8, 133.8,
128.2, 126.0, 121.8, 120.4, 120.2, 108.3, 107.5, 79.7, 37.4, 36.2,
28.0 ppm; IR: n˜ =3402, 1699, 1664, 1557, 1531, 1499, 1468, 1436,
1405, 1386, 1369, 1304, 1273, 1243, 1204, 1165, 1151, 1100, 1084,
1060, 843, 832, 812, 789, 748, 654, 633, 588, 528, 469 cmÀ1; MS
(ESI+): m/z: calcd for C16H20N4O5 +H+: 349.1; found: 349.3 [M+H]+;
elemental analysis calcd (%) for C16H20N4O5: C 55.17, H 5.79, N
16.08; found: C 55.13, H 5.88, N 16.25.
of
5 (1.39 g, 6.13 mmol) in CH2Cl2 (30 mL). DIEA (1.31 mL,
7.67 mmol) was added. After the system had been stirred for 16 h
at room temperature, the solvent was evaporated, and the crude
product was purified by column chromatography (Hex/EtOAc/
CH2Cl2 2:9:1). The product was recrystallized from EtOH to yield 8a
a
colorless solid (1.98 g, 83%). M.p. 128–1298C; 1H NMR
O2N-PyPyPy-OH (10b): Compound 9 (2.00 g, 5.74 mmol) was added
to a suspension of Pd/C (0.20 g, 10% on active charcoal) in anhy-
drous EtOAc (30 mL). The mixture was stirred in a steel autoclave
under H2 (40 bar) for 3 h at 558C. The catalyst was removed by fil-
tration through Celite, and the solvent was evaporated. After
drying of the product in vacuo, the reduced 9 was dissolved in
CH2Cl2 (10 mL). The resulting solution was added dropwise to a
solution of 2 (1.72 g, 6.32 mmol) in CH2Cl2 (30 mL). DIEA (1.47 mL,
8.61 mmol) was added. After the system had been stirred for 16 h
at room temperature, the solvent was evaporated, and the crude
product was purified by column chromatography (Hex/EtOAc 1:1)
as
(250 MHz, [D6]DMSO): d=10.44 (s, 1H; NH), 9.91 (s, 1H; NH), 8.03
(t, J=5.7 Hz, 1H; NH), 7.39 (d, J=0.8 Hz, 1H; Im-H), 7.29 (d, J=
1.7 Hz, 1H; Py-H), 7.18 (d, J=1.7 Hz, 1H; Py-H), 7.16 (d, J=1.9 Hz,
1H; Py-H), 7.04 (d, J=0.8 Hz, 1H; Im-H), 6.88 (d, J=1.9 Hz, 1H; Py-
H), 4.00 (s, 3H; NÀCH3), 3.85 (s, 3H; NÀCH3), 3.80 (s, 3H; NÀCH3),
3.59 (s, 3H; COOCH3), 3.19 (q, J=6.1 Hz, 2H; CH2), 2.34 (t, J=
7.4 Hz, 2H; CH2), 1.74 ppm (quin, J=7.1 Hz, 2H; CH2); 13C NMR
(60 MHz, [D6]DMSO): d=173.1, 161.3, 158.4, 156.1, 138.8, 127.0,
126.3, 123.0, 122.9, 122.0, 121.4, 118.6, 117.8, 104.9, 104.3, 51.2,
37.7, 36.0, 35.8, 35.0, 30.8, 24.6 ppm; IR: n˜ =3407, 3267, 2951, 1716,
1671, 1632, 1578, 1542, 1519, 1467, 1450, 1431, 1403, 1380, 1273,
1255, 1202, 1179, 1145, 1124, 1065, 1010, 979, 937, 883, 813, 785,
764, 736, 699, 660, 639, 620, 548 cmÀ1; MS (ESI+): m/z: calcd for
C22H27N7O5 +H+: 470.2; found: 470.3 [M+H]+, 492.3 [M+Na]+; ele-
mental analysis calcd (%) for C22H27N7O5: C 56.28, H 5.80, N 20.88;
found: C 56.02, H 6.00, N 20.96.
1
to yield 10a as a yellow solid (2.05 g, 75%). M.p. 1788C; H NMR
(250 MHz, [D6]DMSO): d=10.28 (s, 1H; NH), 9.92 (s, 1H; NH), 8.19
(d, J=2.0 Hz, 1H; Py-H), 7.59 (d, J=2.0 Hz, 1H; Py-H), 7.40 (d, J=
2.0 Hz, 1H; Py-H), 7.25 (d, J=1.7 Hz, 1H; Py-H), 7.07 (d, J=1.9 Hz,
1H; Py-H), 6.85 (d, J=2.0 Hz, 1H; Py-H), 3.97 (s, 3H; NÀCH3), 3.86
(s, 3H; NÀCH3), 3.81 (s, 3H; NÀCH3), 1.51 ppm (s, 9H; C(CH3)3);
13C NMR (60 MHz, [D6]DMSO): d=159.9, 158.3, 156.9, 133.8, 128.2,
126.3, 122.8, 122.6, 121.5, 120.2, 120.1, 118.7, 108.4, 107.6, 104.6,
79.6, 37.4, 36.2, 36.1, 28.1 ppm; IR: n˜ =3412, 3372, 3141, 2977,
1715, 1697, 1674, 1645, 1592, 1556, 1522, 1498, 1434, 1422, 1403,
1367, 1315, 1275, 1248, 1216, 1168, 1123, 1102, 1084, 1055, 1008,
Compound 8a (1.00 g, 2.1 mmol) was suspended in a mixture of
MeOH/H2O (3:1, 40 mL) and cooled to 08C. LiOH·H2O (0.44 g,
10.5 mmol) was added, and the mixture was stirred for 16 h.
During this time the solution was allowed to warm up to room
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987, 890, 848, 816, 777, 751, 742, 714, 668, 639, 600, 541 cmÀ1; MS
(ESI+): m/z: calcd for C22H26N6O6 +H+: 471.2; found: 471.8 [M+H]+,
493.9 [M+Na]+; elemental analysis calcd (%) for C22H26N6O6: C
56.16, H 5.57, N 17.86; found: C 56.13, H 5.45, N 18.27.
3064, 2941, 2798, 1690, 1649, 1526, 1454, 1391, 1365, 1342, 1246,
1167, 1138, 1055, 1002, 861, 777, 737, 697, 607, 576, 460 cmÀ1, MS
(ESI+): m/z: calcd for C23H38N4O5 +H+: 451.3; found: 451.4 [M+H]+,
473.2 [M+Na]+; elemental analysis calcd (%) for C23H38N4O5: C
61.31, H 8.50, N 12.43; found: C 61.06, H 8.64, N 12.17.
Compound 10a (2.00 g, 4.25 mmol) was diluted in anhydrous
CH2Cl2 (40 mL). The solution was cooled to 08C, and a solution of
TiCl4 (0.93 mL, 8.50 mmol) in anhydrous CH2Cl2 (15 mL) was added
dropwise. The solution was stirred for a further 30 min at 08C and
was then allowed to warm to room temperature. HCl (1n, 100 mL)
was added slowly, and the solid precipitated. The solid was filtered,
and the residue was washed with MeOH. After drying in vacuo
10b was obtained as a yellow solid (1.43 g, 81%). M.p. 2528C;
1H NMR (250 MHz, [D6]DMSO): d=12.17 (brs, 1H; COOH), 10.32 (s,
1H; NH), 9.96 (s, 1H; NH), 8.19 (d, J=1.7 Hz, 1H; Py-H), 7.61 (d, J=
2.0 Hz, 1H; Py-H), 7.43 (d, J=1.9 Hz, 1H; Py-H), 7.27 (d, J=1.6 Hz,
1H; Py-H), 7.06 (d, J=1.7 Hz, 1H; Py-H), 6.85 (d, J=1.7 Hz, 1H; Py-
H), 3.96 (s, 3H; NÀCH3), 3.86 (s, 3H; NÀCH3), 3.82 ppm (s, 3H; NÀ
CH3); 13C NMR (60 MHz, [D6]DMSO): d=158.3, 156.9, 133.8, 128.2,
126.3, 123.1, 122.2, 121.4, 118.5, 107.6, 107.5, 104.5, 37.4, 36.1,
36.0 ppm; IR: n˜ =3410, 3379, 3138, 3110, 2924, 2853, 1674, 1645,
1591, 1560, 1522, 1498, 1435, 1420, 1402, 1355, 1309, 1257, 1210,
1174, 1153, 1123, 1110, 1062, 1005, 988, 889, 856, 834, 816, 779,
750, 738, 709, 666, 645, 591, 565, 520, 466 cmÀ1; MS (ESIÀ): m/z:
calcd for C18H18N6O6ÀH+: 413.1; found: 413.7 [MÀH]+.
Cbz-b-Ala-C3-N(Me)-C3-GABA-NHBoc (14): Dry MeOH (5 mL) was
cooled to 08C, and acetyl chloride (1 mL) was slowly added drop-
wise, followed by stirring for 30 min. Compound 13 (1.50 g,
3.33 mmol) was dissolved in anhydrous MeOH (5 mL) and added
dropwise to the acidic MeOH solution, and the mixture was stirred
at 08C for 30 min, followed by 60 min at ambient temperature.
When the reaction was complete, the solvent was evaporated, and
the residue was dried in vacuo. The deprotected 13, Boc-GABA
(0.81 g, 3.99 mmol), DIEA (2.28 mL, 13.32 mmol), and HOBt (0.62 g,
3.99 mmol) were dissolved in anhydrous DMF (10 mL) and warmed
up to 608C. After that temperature had been reached, DIC
(1.03 mL, 6.66 mmol) was added, and the resulting solution was
stirred for 4 h at 608C. The solution was allowed cool to room tem-
perature and was then slowly added dropwise to a solution of sat.
NaHCO3/H2O (1:4, 50 mL). The resulting yellow solution was ex-
tracted three times with CH2Cl2. The combined organic layer was
washed with water and dried (MgSO4). The solvent was evaporat-
ed, and the crude product was purified by column chromatogra-
phy (EtOAc/MeOH 6:1+1% NEt3) to yield 14 as a colorless oil
(1.54 g, 87%); 1H NMR (250 MHz, CDCl3): d=7.40–7.30 (m, 5H;
phenyl), 7.17 (brs, 1H; NH), 7.03 (brs, 1H; NH), 5.72 (brs, 1H; NH),
5.08 (s, 2H; benzyl-CH2), 5.00 (brs, 1H; NH), 3.47 (q, J=6.2 Hz, 2H;
CH2), 3.30 (q, J=6.1 Hz, 4H; 2CH2), 3.12 (q, J=6.3 Hz, 2H; CH2),
2.46–2.38 (m, 6H; CH2), 2.23–2.17 (m, 5H; CH3 and CH2), 1.77 (quin,
J=7.0 Hz, 2H; CH2), 1.66 (quin, J=6.4 Hz, 4H; CH2), 1.42 ppm (s,
9H; C(CH3)3); 13C NMR (60 MHz, CDCl3): d=172.8, 171.4, 156.6,
156.5, 136.6, 128.4, 128.0, 127.9, 77.3, 66.5, 55.7, 55.6, 41.6, 39.9,
38.0, 37.4, 36.0, 33.8, 28.4, 26.7, 26.4 ppm. IR: n˜ =3297, 3063, 2935,
2798, 1684, 1640, 1533, 1453, 1390, 1365, 1341, 1267, 1239, 1168,
1140, 1108, 1028, 1001, 910, 874, 851, 779, 750, 729, 697, 630, 618,
576, 465 cmÀ1; HRMS (MALDI): m/z: calcd for C27H45N5O6 +H+:
536.34426; found: 536.3437 [M+H]+.
3-(tert-Butyloxycarbonylaminopropyl)-3’-aminopropyl-N-methylamine
(12): Compound 11 (29.55 mL, 183 mmol) was dissolved in 1,4-di-
oxane (50 mL). A solution of Boc2O (10.00 g, 45.8 mmol) in 1,4-diox-
ane (50 mL) was added dropwise to the resulting solution over 2 h.
The colorless suspension was stirred overnight. Afterwards the sol-
vent was evaporated; the residue was redissolved in water and ex-
tracted with CH2Cl2. The combined organic layer was washed with
water and dried over MgSO4, and the solvent was evaporated. The
crude product was distilled in vacuo (1 mbar, 2008C) to afford a col-
1
orless oil in 79% yield (8.84 g); H NMR (250 MHz, CDCl3): d=5.47
(brs, 1H; NH), 3.13 (q, J=6.0 Hz, 2H; NHCH2), 2.70 (t, J=6.9 Hz,
2H; NH2CH2), 2.36–2.30 (m, 4H; 2CH2), 2.14 (s, 3H; NÀCH3), 1.64–
1.54 (m, 6H; CH2N(Me)CH2, NH2), 1.39 ppm (s, 9H; C(CH3)3);
13C NMR (60 MHz, [D6]DMSO): d=155.5, 77.0, 55.1, 54.9, 41.7, 40.0,
38.3, 30.9, 28.1, 27.2 ppm, IR: n˜ =3356, 2974, 2933, 2866, 2793,
1693, 1517, 1454, 1390, 1364, 1273, 1249, 1168, 1072, 1047, 1002,
971, 861, 778, 643, 461 cmÀ1; HRMS (MALDI): m/z: calcd for
C12H27N3O2 +H+: 246.21760; found: 246.2178 [M+H]+.
O2N-PyPyPy-b-Ala-C3-N(Me)-C3-GABA-NHBoc (15): Compound 14
(1.00 g, 2.22 mmol) was added to a suspension of Pd/C (0.30 g,
10% on active charcoal) in anhydrous MeOH (20 mL). The mixture
was stirred in a steel autoclave under H2 (40 bar) for 3 h at 558C.
The catalyst was removed by filtration through Celite, and the sol-
vent was evaporated. After drying of the product in vacuo, the
product was dissolved in anhydrous DMF. The resulting solution
was added dropwise to a solution of 10b (1.24 g, 2.99 mmol) in
abs. DMF (10 mL), followed by DIEA (1.28 mL, 7.47 mmol) and
HOBt (0.46 g, 2.99 mmol). The solution was heated to 608C, and
DIC (0.77 mL, 4.98 mmol) was added. The solution was stirred for
4 h at 608C. Afterwards the solution was added to a mixture of sat.
NaHCO3/water (1:4, 50 mL). The layers were separated, and the
aqueous layer was extracted with CH2Cl2. The combined organic
layer was dried over MgSO4, and the solvent was evaporated. The
crude product was purified by column chromatography (CH2Cl2/
MeOH 9:1+1% NEt3) to yield 15 as a yellow foam (1.28 g, 72%).
1H NMR (250 MHz, [D6]DMSO): d=10.31 (s, 1H; NH), 9.95 (s, 1H;
NH), 8.18 (d, J=1.6 Hz, 1H; Py-H), 8.00 (t, J=5.5 Hz, 1H; NH), 7.90
(t, J=5.5 Hz, 1H; NH), 7.79 (t, J=5.5 Hz, 1H; NH), 7.61 (d, J=
2.1 Hz, 1H; Py-H), 7.28 (d, J=1.7 Hz, 1H; Py-H), 7.20 (d, J=1.6 Hz,
1H; Py-H), 7.04 (d, J=1.8 Hz, 1H; Py-H), 6.84 (d, J=1.7 Hz, 1H; Py-
H), 6.77 (t, J=5.4 Hz, 1H; NH), 3.97 (s, 3H; NÀCH3), 3.86 (s, 3H; NÀ
CH3), 3.81 (s, 3H; NÀCH3), 3.37 (q, J=6.7 Hz, 4H; CH2), 3.08–3.05
(m, 4H; CH2), 2.88 (q, J=6.5 Hz, 2H; CH2), 2.36–2.28 (m, 4H; CH2),
Cbz-b-Ala-C3-N(Me)-C3-NHBoc (13): Compound 12 (3.00 g,
12.23 mmol), Cbz-b-Ala-OH (3.28 g, 14.67 mmol), DIEA (4.19 mL,
24.45 mmol), and HOBt (2.26 g, 14.67 mmol) were dissolved in an-
hydrous DMF (20 mL) and warmed to 608C. After that temperature
had been reached, DIC (3.79 mL, 24.45 mmol) was added, and the
brown solution was stirred for 4 h. The solution was allowed to
cool to room temperature and was then slowly added dropwise to
a solution of sat. NaHCO3/H2O (1:4, 100 mL). The resulting yellow
solution was extracted three times with CH2Cl2, and the combined
organic layer was washed with water and dried (MgSO4). The sol-
vent was evaporated, and the crude product was purified by
column chromatography (EtOAc/MeOH 6:1+1% NEt3) to yield 13
1
as a colorless oil (4.81 g, 87%); H NMR (250 MHz, CDCl3): d=7.26–
7.22 (m, 5H; benzyl-H), 7.12 (brs, 1H; NH), 5.67 (brs, 1H; NH), 5.11
(brs, 1H; NH), 5.00 (s, 2H; benzyl-CH2), 3.39 (q, J=6.1 Hz, 2H; CH2),
3.23 (q, J=6.0 Hz, 2H; CH2), 3.08 (q, J=5.5 Hz, 2H; NHCH2), 2.35–
2.26 (m, 6H; CH2CH2CH2), 2.09 (s, 3H; NÀCH3), 1.59–1.50 (m, 4H;
CH2N(Me)CH2), 1.35 ppm (s, 9H; C(CH3)3); 13C NMR (60 MHz, CDCl3):
d=171.2, 156.4, 156.1, 136.6, 128.3, 127.9, 127.8, 79.0, 66.4, 55.8,
55.5, 41.6, 38.9, 38.3, 37.3, 35.9, 28.3, 27.4, 26.0 ppm; IR: n˜ =3320,
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2.13 (s, 3H; NÀCH3), 2.03 (t, J=7.7 Hz, 2H; CH2), 1.63–1.49 (m, 6H;
CH2), 1.36 ppm (s, 9H; C(CH3)3); 13C NMR (60 MHz, [D6]DMSO): d=
171.6, 170.4, 161.2, 158.3, 156.9,155.5, 133.8, 128.1, 126.3, 123.0,
122.8, 122.1, 121.4, 118.6, 117.9, 107.6, 104.5, 104.1, 77.4, 54.6, 52.0,
48.5, 45.6, 41.4, 37.4, 36.7, 36.1, 35.9, 35.5, 35.4, 32.9, 28.2, 26.6,
25.8 ppm; IR: n˜ =3288, 2936, 1641, 1525, 1505, 1464, 1435, 1398,
1364, 1308, 1252, 1204, 1165, 1099, 1061, 1005, 886, 813, 775, 750,
711, 667, 596, 467 cmÀ1; HRMS: m/z: calcd for C37H55N11O9 +H+:
798.42570; found: 798.42645 [M+H]+.
with CH2Cl2. The combined organic layers were dried over MgSO4,
and the solvent was evaporated. The crude product was purified
by column chromatography (EtOAc/MeOH 1:1+1% NEt3) to afford
18 as an off-white foam (210 mg, 62%). MS (ESI+): m/z: calcd for
C82H104N20O16 +H+: 1625.8; found: 1625.2 [M+H]+.
Im-Py-Py-GABA-PyPyPy-b-Ala-C3-N(Me)-C3-GABA-NH-bis(guanidinium)-
alcohol (1): Dry MeOH (2 mL) was cooled to 08C, and acetyl chlo-
ride (500 mL) was slowly added dropwise, followed by stirring for
30 min. Compound 18 (60 mg, 37 mmol) was dissolved in anhy-
drous MeOH (1 mL), added dropwise to the acidic MeOH solution,
and stirred at 08C for 30 min, followed by 60 min at ambient tem-
perature. When the reaction was complete, the solvent was evapo-
rated, and the residue was dried in vacuo. The deprotected amine
was dissolved in a MeOH/NEt3 mixture (2:1, 6 mL). 4,5-Dihydroimi-
dazole-2-sulfonic acid (22 mg, 0.149 mmol) was added, and the re-
sulting solution was stirred for 16 h. After HPLC purification (Repro-
sil AQ C18 5 mm, 250 mm, isocratic, 0.1% aqueous TFA+acetonitrile
74:26, 9 mLminÀ1), 1 was obtained as a yellowish foam (11.2 mg,
19%). MS (ESI+): m/z: calcd for C78H96N24O12 +H+: 1561.8; found:
1561.2 [M+H]+.
Im-Py-Py-GABA-PyPyPy-b-Ala-C3-N(Me)-C3-GABA-NH2 (16): Compound
15 (0.25 g, 0.31 mmol) was added to a suspension of Pd/C (0.10 g,
10% on active charcoal) in anhydrous MeOH (20 mL). The mixture
was stirred in a steel autoclave under H2 (40 bar) for 2 h at 508C.
The catalyst was removed by filtration through Celite, and the sol-
vent was evaporated. After drying of the product in vacuo, the
product was dissolved in anhydrous DMF. The resulting solution
was added dropwise to a solution of 8b (0.16 g, 0.35 mmol) in abs.
DMF (5 mL), followed by DIEA (0.07 mL, 0.41 mmol) and HOBt
(0.06 g, 0.39 mmol). The solution was heated to 608C, and DIC
(0.06 mL, 0.40 mmol) was added. The solution was stirred for 3 h at
608C. Afterwards the solution was added to a sat. NaHCO3/water
mixture (1:4, 25 mL). The layers were separated, and the aqueous
layer was extracted with CH2Cl2. The combined organic layer was
dried over MgSO4, and the solvent was evaporated. The crude
product was purified by column chromatography (EtOAc/MeOH
1:1+1% NEt3). Dry MeOH (1 mL) was cooled to 08C, and acetyl
chloride (50 mL) was slowly added dropwise, followed by stirring
for 30 min. Boc-protected 16 (0.21 g, 0.17 mmol) was dissolved in
anhydrous MeOH (1 mL), added dropwise to the acidic MeOH solu-
tion, and stirred at 08C for 30 min, followed by 60 min at ambient
temperature. When the reaction was complete, the solvent was
evaporated, and the residue was dried in vacuo. Compound 16
DNA duplex stabilization by compound 1: All experiments were
executed with the same 11-mer DNA duplex as previously reported
for study of the interaction with hairpin polyamide 19: d(CATTGT-
TAGAC)3’:d(GTCTAACAATG)3’.[18] UV: JASCO V-650, conditions:
sodium cacodylate (pH 7.0, 10 mm), KCl (10 mm), MgCl2 (10 mm),
CaCl2 (5 mm); profiles were measured at 260 nm, heating rate
18CminÀ1, 3 cycles, duplex (5 mm) and 1 (5 mm). CD titrations:
JASCO AKS J-810, conditions: sodium cacodylate (pH 7.0, 10 mm),
KCl (10 mm), MgCl2 (10 mm), CaCl2 (5 mm), 208C, duplex (5 mm),
and incremental addition of 1.
DNA cleavage assay: Plasmid preparation, sample incubation, and
electrophoretic analysis followed the previously published meth-
ods exactly.[14b]
1
was obtained as a yellowish foam (0.15 g, 43%). H NMR (300 MHz,
[D6]DMSO): d=10.47 (s, 1H; NH), 9.91–9.84 (m, 3H; NH), 9.42 (brs,
1H; NH), 8.07–8.05 (m, 4H; NH), 7.79 (brs, 3H; NH3), 7.42–6.88 (m,
12H; Py-H, Im-H), 4.00 (s, 3H; CH3), 3.85–3.80 (m, 15H; CH3), 3.39–
3.37 (m, 2H; CH2), 3.25–3.19 (m, 2H; CH2), 3.11–3.00 (m, 8H; CH2),
2.80–2.73 (m, 5H; CH2, CH3), 2.38–2.26 (m, 4H; CH2), 2.18 (t, J=
7.2 Hz, 2H; CH2), 1.77–1.73 ppm (m, 8H; CH2); 13C NMR (75 MHz,
[D6]DMSO): d=171.53, 171.01, 169.26, 161.31, 161.28, 158.46,
158.01, 155.77, 138.66, 126.56, 126.32, 123.07, 122.95, 122.77,
122.71, 122.17, 122.13, 122.07, 122.05, 121.35, 118.64, 118.49,
118.45, 118.15, 117.94, 117.83, 104.90, 104.29, 53.07, 38.59, 36.10,
36.05, 35.93, 35.70, 35.62, 35.58, 35.14, 33.30, 31.92, 25.70, 24.03,
23.96, 23.06 ppm; IR: n˜ =3283, 3088, 2927, 1634, 1575, 1532, 1464,
1435, 1403, 1259, 1176, 1128, 1062, 1005, 833, 797, 777, 720, 706,
Acknowledgements
Financial support by the European 7th framework program
(238679, PhosChemRec) is gratefully acknowledged.
Keywords: DNA cleavage · enzyme models · guanidine ·
minor groove binders · nucleic acids
668, 606, 518, 478, 465 cmÀ1
;
MS (ESI+): m/z: calcd for
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C53H72N18O9 +H+: 1105.9; found: 1106.6 [M+H]+.
[2] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, E. Charpentier,
Science 2012, 337, 816–821.
[3] Y. Aiba, J. Sumaoka, M. Komiyama, Chem. Soc. Rev. 2011, 40, 5657–
5668.
Im-Py-Py-GABA-PyPyPy-b-Ala-C3-N(Me)-C3-GABA-NH-bis(tert-butoxy-
carbonylamino)alcohol (18): Acetyl chloride (2 mL) was slowly
mixed at 08C with dry MeOH (5 mL). After the mixture had been
kept at 08C for 30 min, a solution of Boc-protected 16 (250 mg,
0.207 mmol) in dry MeOH (1 mL) was added, and the mixture was
stirred for 30 min at 08C and for 30 min at room temperature.
After removal of the solvent and drying in vacuo, the residue of 16
was dissolved in anhydrous DMF (1 mL) and added to a solution of
17 (130 mg, 0.241 mmol), DIEA (0.64 mL, 3.73 mmol), and HOBt
(40 mg, 0.26 mmol) in anhydrous DMF (4 mL). The solution was
heated to 608C, and DIC (80 mL, 0.52 mmol) was added. The solu-
tion was stirred for 3 h at 608C. Afterwards it was added to a mix-
ture of sat. NaHCO3/water (1:4, 50 mL) and CH2Cl2 (20 mL). The
layers were separated, and the aqueous layer was extracted twice
[4] a) T. Gaj, C. A. Gersbach, C. F. Barbas III, Trends Biotechnol. 2013, 31,
397–405; b) Y. G. Kim, J. Cha, S. Chandrasegaran, Proc. Natl. Acad. Sci.
USA 1996, 93, 1156–1160; c) J. Bitinaite, D. A. Wah, A. K. Aggarwal, I.
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Manuscript received: October 22, 2015
Accepted article published: January 8, 2016
Final article published: February 16, 2016
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