U-pin polyamide motif for recognition of the DNA minor groove

Article abstract of DOI:10.1002/chem.200304784

DNA-binding hairpin pyrrole-imidazole polyamides with γ-aminobutyric acid as a turn-forming residue tolerate A · T or T · A base pairs under the turn. U-pins - polyamides with a different turn - have been synthesized and their DNA binding properties were studied. The two turn-forming residues are connected via the ring nitrogens using variable length aliphatic linkers ((CH₂)_n, n = 3-6). Through optimization of the linker length and the substituents at the 2-position of the pyrrole residue on the U-turn, polyamides with G · C/C · G tolerant turns could be found, which bind to DNA in a predictable manner.

Full text of DOI:10.1002/chem.200304784

FULL PAPER
U-Pin Polyamide Motif for Recognition of the DNA Minor Groove
Alexander Heckel and Peter B. Dervan*[a]
Abstract: DNA-binding hairpin pyrrole ± imidazole polyamides with g-aminobuty-
ric acid as a turn-forming residue tolerate A ¥ T or T¥ A base pairs under the turn.
U-pins–polyamides with a different turn–have been synthesized and their DNA
binding properties were studied. The two turn-forming residues are connected via the
ring nitrogens using variable length aliphatic linkers ((CH ) , n ˆ 3 ± 6). Through
Keywords: amino acids
recognition pyrrole ± imidazole
polyamides ¥ U-pins
¥ DNA
¥
2
n
optimization of the linker length and the substituents at the 2-position of the pyrrole
residue on the U-turn, polyamides with G ¥ C/C ¥ G tolerant turns could be found,
which bind to DNA in a predictable manner.
Introduction
high affinity and selectivity; however, there are limitations.
Covalently linking the antiparallel polyamide strands with a
g-aminobutyric acid residue (GABA) in the hairpin motif
affords higher affinity binders. This also ensures a heterodi-
meric[ binding mode with the DNA and locks the register of
the two strands with respect to each other, increasing the
specificity.[ However, the hairpin motif has a sequence
requirement under the GABA turn residue, tolerating only
A ¥ T/T¥ A base pairs for steric reasons.[ In a recent inves-
tigation the influence of different tail residues on G/C
tolerance was studied. Here, the ability of a new turn motif
to provide a similar G/C tolerance at the other end of the
recognition site are explored.
The intolerance of a GABA turn toward G ¥ C/C ¥ G base
pairs is due to the conflict with the steric bulkpresented by the
Polyamides made from N-methylpyrrole (Py), N-methylimi-
dazole (Im) and N-methyl-3-hydroxypyrrole (Hp) amino
acids bind with high affinities and sequence specificities to
the minor groove of double stranded DNA. This opens the
door to an array of potential applications, for example
regulating gene expression and chromosomal staining.[
The progression from the natural products netropsin and
distamycin to the current picture of DNA recognition with
polyamides has been summarized in a recent review article.[1a]
This journey has included systematic variations of atoms,
substituents, residues and entire motifs and the DNA-binding
characteristics of each variant have been measured by
quantitative footprinting titrations. Hairpin polyamides con-
sist of two strands of aromatic amino acids which are
positioned antiparallel side-by-side in the minor groove of
DNA. A pairing rule paradigm has been developed, wherein a
Py/Im pair targets a C ¥ G base pair, while an Im/Py pair
targets a G ¥ C base pair. Likewise, a Py/Hp combination
distinguishes A ¥ T from T¥ A. A Py/Py pair is degenerate and
binds to both A ¥ T and T¥ A but not to G ¥ C or C ¥ G.
Figure 1[ shows a mnemonic to illustrate how polyamides
read the recognition pattern present in the minor groove. This
model presents our view of polyamides as modular DNA-
recognition tools, constructed using simple rules and a small
set of readily available monomer units (the residues). Poly-
amides target a considerable number of DNA sequences with
4]
5]
1, 2]
6]
[7]
exocyclic amino group of a guanine residue. This NH group
2
points outwards from the bottom of the minor groove ™on∫
which the alkyl chain of the GABA turn lies. An alternative
way to connect two polyamide strands is the ™H-pin∫ motif[
in which two N-methyl groups of central residues are
connected with an alkyl chain leading to polyamide molecules
that resemble the letter ™H∫ (Figure 2). While these mole-
cules have the potential to target G ¥ C/C ¥ G tracts they are
significantly different in their architecture from hairpin
polyamides because they have a branched structure. As a
result they are more difficult to synthesize especially on solid
phase.[ Moving the bridge away from the internal residues,
toward the edge of the molecule creates a linear oligomer
resembling the letter ™U∫ (Figures 2 and 3).
8, 9]
3]
9]
[
a] Prof. P. B. Dervan, Dr. A. Heckel
California Institute of Technology
Division of Chemistry and Chemical Engineering
The Beckman Institute
We refer to this new class of polyamides as ™U-pins∫ and to
the turn as a ™U-turn∫. While this only seems to be a minor
change, there were some caveats: To tolerate adjacent G/C
base pairs, it was possible that the substituent R (Figure 3) on
the U-pin would have to be hydrogen, to avoid a steric clash.
Compared with the analogous hairpin polyamide 2, such a
Pasadena, CA 91125 (USA)
Fax : (‡1)626-683-8754
E-mail: dervan@caltech.edu
Chem. Eur. J. 2003, 9, 3353 ± 3366
DOI: 10.1002/chem.200304784
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3353

P. B. Dervan and A. Heckel
FULL PAPER
Results and Discussion
To prepare U-pins using estab-
lished solid-phase synthesis
methods,[ it was necessary to
11]
synthesize
U-turn
dimers
(
Scheme 1). Starting from the
commercially available substi-
tuted pyrrole 3 compound 6 was
obtained after nitration (!4),
esterification (!5), reduction
and Boc protection. Then imi-
dazole was alkylated with ace-
tyl-protected bromo alcohols of
different chain lengths to ob-
tain compounds 7a ± c. After
tricholoroacetylation the result-
ing trichloroketones 8a ± c were
converted to the O-deprotected
alcohols 9a ± c. To obtain the
dimer the alcohols were con-
verted to the respective iodides
using I , PPh and imidazole.
Figure 1. Dip-and-bump-mnemonic for polyamides reading the recognition pattern of the DNA minor groove.
The pairing rules are represented as shape complementarities. Pyrrole residues are drawn in yellow, imidazole
residues in red and hydroxypyrrole residues in blue. The bottom of the minor groove (shown as a crescent)
presents its recognition pattern as a series of dips and bumps ± dips for hydrogen-bond acceptors and bumps for
hydrogen bond donors. A Py residue can be paired with itself (this combination tolerates but does not
discriminate between A ¥ Tand T¥ A base pairs) or with an Im or an Hp residue. The former is drawn as a red ball
2
3
After aqueous workup the
crude mixture of the iodide
and OPPh3 was directly used
for the alkylation of compound
2
with a dip (a hydrogen bond acceptor position) matching the bump (the NH group) of a guanine base, the latter
as a blue ball with a bump symbolizing the steric bulkand hydrogen bond donor capability of the hydroxy group.
For polyamides with good binding affinities the following two rules must be obeyed: All bumps have to be
matched by a dip and vice versa. As only exception, a Py/Py pair with its neutral surface can match the dip of an
[
3]
A ¥ T base pair.
6
1
to provide the compounds
0a ± c. This step was followed
by deprotection of the carbox-
ylic acid with TBAF and formation of methyl amides 11a ± c.
Finally, the building blocks 12a ± c were obtained after
saponification of the ethyl ester. Because compounds 12a ± c
readily decarboxylate upon acidification they were used as
their sodium salts.
The six-carbon bridged dimers 12d, 16 and 20 required a
slightly different route (Scheme 2). Since the requisite acetyl-
protected bromo alcohol is not commercially available, 1,6-
hexanediol as used as the starting material. After monoace-
tylation[ (!13) the protected diol was treated with I , PPh
12]
2
3
Figure 2. Three motifs for DNA-binding polyamides: Hairpin, U-pin and
H-pin. As opposed to the two former, the latter has a branched architecture
with a concomitant additional level of complication in the synthesis. In this
black-and-white pictogram representation imidazole residues are drawn as
blackcircles and pyrrole residues as open circles. A methyl amide tail is
drawn as a triangle, a b-alanine residue as a diamond and an aminopropyl
group as a crescent with a ™‡∫ sign. For structural formulae of these
polyamides see Figure 3 and ref. [9]. The arrows indicate the N ! C
direction in the polyamide.
and imidazole. The crude iodide obtained after aqueous
workup was used directly for the alkylation of imidazole
(
!7d). Trichloroacetylation (!8d) and esterification afford-
ed compound 9d, which was converted to the iodide and
treated with the nitro-substituted pyrrole 5 to obtain com-
pound 14. Nitropyrrole 5 was found to give better alkylation
yields than Boc-aminopyrrole 6. Reduction and Boc protec-
tion of compound 14 provided compound 10d. Deprotection
and amide bond formation using MeNH or NH afforded
2
3
U-pin would have two fewer hydrogen bonds, and potentially
lower affinity. Furthermore, U-pins could theoretically bind to
DNA in modes other than the predicted ™normal∫ one
compounds 11d and 15, respectively. After saponification, the
carboxylic acid sodium salts 12d and 16 were obtained and
again used without acidification to avoid decarboxylation.
Preparing the minimally substituted component 18 using the
(
Figure 4). U-pins could bind in reverse fashion, as is observed
[10]
[13]
for certain hairpin polyamides.
crude iodide 17 and 3-nitropyrrole proved to be impractical,
due to separation difficulties–the use of purified iodide 17,[
however, proceeded smoothly. After reduction and Boc
protection (!19), saponification afforded the sodium salt
20. The remaining building blocks for the solid-phase syn-
thesis were either commercially available or obtained accord-
14]
Additionally, U-pins could extend and bind in a ™Z binding
mode∫ or, if the bridge were long enough, in a fully linear
binding mode. To address these questions, the U-pins 1a ± f
which have different linker lengths and substituents were
synthesized.
3354
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2003, 9, 3353 ± 3366

U-Pin Polyamide Motif
3353±3366
ing
dures.[
to
literature
Following estab-
proce-
2, 15, 16]
lished solid phase synthesis pro-
tocols[
7, 11]
and using oxime res-
in, the U-pins 1a ± f and the
hairpin 2 were synthesized and
purified
Scheme 3).
At first the question of the
by
RP-HPLC
(
optimal linker length was ad-
dressed. Therefore, DNase I
footprinting titration experi-
ments were performed with
the U-pins 1a ± d and the hair-
pin polyamide 2 as reference
compound on a restriction frag-
ment of the plasmid pDEH9
(
Figure 5). This 250 base-pair
sequence has several potential
binding sites of interest. Ideally,
the U-pins 1a ± f should read a
sequence 5'-GGWC-3' (Wˆ
A,T). The site labeled ™A∫ is
the only match site on this
restriction fragment; sites ™B∫
and ™C∫ are both designed
single base pair mismatch sites.
Site ™D∫ contains overlapping
Figure 3. A comparison between U-pins and hairpins. Right: The U-pins 1a ± f and the reference hairpin
polyamide 2 synthesized for this study. Left: Hydrogen bonding pattern of hairpins and–extrapolating–the
putative hydrogen bonding pattern of U-pins. In this ™exploded view∫ the minor groove of DNA is drawn as
ladder. The steps of the ladder are the planes of the base pairs which present their respective recognition
elements: electron pairs (two dots in a circle) and H-bond donors (H in a circle).
regions to which the U-pins
could bind in all of the unwant-
ed binding modes (see Fig-
ure 4). The lower left corner of
Figure 5 shows the result of a
footprinting titration with the
+
+
5
'
3'
5'
5'
3'
3'
5'
reference hairpin 2. The affinities to the respective binding
sites are summarized in Table 1. Reference hairpin 2 binds
with high affinity and specificity to the match site
(site ™A∫).
3
'
normal binding mode
+
reverse binding mode
Surprisingly, the U-pins showed a very similar behavior. In
all cases the footprint at the match binding site is the
dominant feature. U-pins 1a, b and d bind the DNA with
affinities nearly equal to that of reference compound 2–a
surprising result since these U-pins have one hydrogen bond
less than the reference compound. Only U-pin 1c with a five-
carbon linker has a significantly lower affinity in this DNA
context. A recent study on H-pins[ showed a similar behavior
for the linker lengths: polyamides with a 4- or 6-carbon bridge
displayed the highest affinities, while polyamides with a five-
carbon bridge displayed lower affinities.
5'
3'
5'
5'
3'
5'
+
3'
3'
Z binding modes
+
9]
5
'
3'
5'
3'
linear binding mode
Figure 4. Representations of theoretically possible binding modes of
U-pins. In the ™normal∫ binding mode the N ! C direction of the
polyamide is parallel to the 5' ! 3' direction of the DNA, whereas in the
reverse binding mode it is antiparallel. These two binding modes have been
observed for hairpin polyamides with the latter occurring in some
Significantly, the U-pins bind DNA in the ™normal∫ mode
essentially exclusively. For example, no footprint is observed
at site D, where the U-pins could theoretically bind in several
possible undesired binding modes. Furthermore, the U-pins
display specificities for the match over the single base pair
mismatch site that are comparable to the ones of hairpin
polyamide 2. Non-specific binding was only observed with
U-pin 1d at 5 nm or higher concentrations. Nonetheless, U-pin
[
10]
exceptional cases only. Depending on the length of the bridge in the
U-turn, binding in a previously unknown ™Z binding mode∫ or–in case of
longer bridges–in
a linear (or 1:1) binding mode could also be
theoretically possible. Since the upper and lower strands of the DNA are
arbitrarily defined, the polyamide could of course also fit into the minor
groove after a 1808 rotation around an axis perpendicular to the helix
axis–given an appropriate recognition pattern in the minor groove.
1
d displays significant DNA binding at concentrations below
5 nm and it had the best overall affinity in the series.
Chem. Eur. J. 2003, 9, 3353 ± 3366
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3355

P. B. Dervan and A. Heckel
FULL PAPER
O2N
O2N
BocHN
a)
b)
c)
CCl3
CCl3
OAc
OTMSE
OTMSE
N
H
N
N
N
O
H
O
H
O
H
O
3
4
5
6
OAc
(CH2)n
N
OH
(CH2)n
H
(CH2)n
N
e)
O
f)
O
d)
N
N
N
Cl3C
EtO
N
N
N
7
a-c
8a-c
(n=3-5)
9a-c
(n=3-5)
(
n=3-5)
BocHN
O
BocHN
O
BocHN
OTMSE
NHMe
NHMe
N
N
N
g) h)
i) j)
k)
O
O
O
(CH2)n
(CH2)n
(CH2)n
O
N
N
N
EtO
EtO
NaO
N
N
N
1
0a-c
11a-c
(n=3-5)
12a-c
(n=3-5)
(
n=3-5)
Scheme 1. Synthesis of the U-turn building blocks 12a ± c. a) HNO
3
, Ac
2
O; b) TMSEOH, NaH; c) 1. H
2
, Pd/C, 2. Boc
2
3 2 n
O, NaHCO ; d) Br(CH ) OAc, NaH;
e) CCl
3
COCl; f) NaOEt; g) I
2
, PPh
3
, Im; h) 6, K
2
CO
3
; i) TBAF; j) 1. DCC, HOBt, 2. MeNH
2
, DIEA; k) NaOH.
OAc
OAc
OH
(CH2)6
N
(CH2)6
O
(CH2)6
N
O
a)
b) c)
d)
e)
N
HO (CH2)6 OH
HO (CH2)6 OAc
Cl3C
EtO
13
N
N
N
7d
8d
9d
O2N
BocHN
BocHN
O
BocHN
O
OTMSE
OTMSE
R
R
N
N
N
N
b) f)
g)
h) i)
j)
O
O
O
O
(CH2)6
(CH2)6
N
(CH2)6
N
(CH2)6
N
O
O
N
EtO
EtO
EtO
NaO
N
N
N
N
1
1d: R=NHMe
12d: R=NHMe
16: R=NH2
14
10d
1
5: R=NH2
O2N
BocHN
BocHN
I
O
(CH2)6
N
N
N
N
b)
k)
g)
j)
9d
(CH2)6
N
(CH )
(CH )
2 6
2 6
EtO
O
O
O
N
N
N
EtO
EtO
NaO
17
N
N
N
18
19
20
Scheme 2. Synthesis of the U-turn building blocks 12d, 16 and 20. a) Ce(SO
CO ; g) H , Pd/C, Boc O; h) TBAF; i) 1. DCC, HOBt, 2. MeNH or NH
4
)
2
/silica gel, EtOAc; b) I
2 3 3
, PPh , Im; c) Im, NaH; d) Cl CCOCl; e) NaOEt; f) 5,
[
13]
K
2
3
2
2
2
3
, DIEA; j) NaOH; k) 3-nitropyrrole,
2 3
K CO .
The tolerance for G/C adjacent to the U-turn element was
binding affinity drops by one order of magnitude for X ˆ C
and no binding was observed for X ˆ G in the concentration
ranges used. This confirms the trend which had been
previously found for hairpin polyamides.[ U-pins 1b and d
(with methyl amide substituents) encounter a lower energetic
penalty for binding the sites with X ˆ C or G, but these
compounds also bind with slightly lower affinities than hairpin
polyamide 2. As pointed out earlier, a U-pin, such as 1b or d,
has one hydrogen bond less than a comparable hairpin
polyamide such as 2. Interestingly, in this DNA context U-pin
1b showed approximately the same affinity as U-pin 1d, while
U-pin 1d showed a higher affinity on the restriction fragment
examined using plasmid pAH2 which incorporates designed
sites 5'-TGGTCX-3' that differ only in the base pair adjacent
to the core binding site (Figure 6). The X ˆ T site appears
twice because it is also part of the plasmid pUC19,[ from
which pAH2 was cloned.
6]
17]
Figure 7 shows the results of the footprinting experiments
with reference hairpin polyamide 2 and U-pins 1d ± f. The
binding affinities to the respective sites are summarized in
Table 2 and represented graphically in Figure 8.
As expected, the GABA residue of the hairpin polyamide 2
tolerates an A ¥ Tor T¥ A base pair at the turn position, but the
3356
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3353 ± 3366

U-Pin Polyamide Motif
3353±3366
O
O
BocHN
N
NO2
PhthN
N
OBt
OH
O
R
O
BocHN
BocHN
P
BocHN
N
OH
OH
N
(
CH2)n
21
O
oxime resin
OH
23
N
NaO
N
BocHN
a) b) c) b) c)
Boc
N
24
O
N
12a-d, 16, 20
N
OLi
2
2
P
N
b) e) f) g) b) c)
Boc
25
b) d)
Boc
R
n
P
P
P
b) h) b) e) f) i)
NPhth
b) h) b) e) f) i)
NPhth
R
n
P
j)
j)
H3N
O
H3N
O
O
CF3COO
N
CF3COO
N
O
R
O
NH
O
N
N
(
CH2)n
N
H
N
H
H
N
N
N
O
O
N
N
N
N
N
H
N
H
H
H
N
N
N
N
N
N
N
N
H
H
N
H
H
N
N
O
N
O
N
N
H
H
N
O
N
O
N
N
H
H
N
N
O
N
O
O
N
2
O
1a-f
Scheme 3. Solid-phase synthesis of the U-pin polyamides 1a ± f and hairpin polyamide 2 on oxime resin. In the pictogram representation the linker of the
oxime resin is drawn as a gray bar and the pyrrole and imidazole residues as circles according to the rules explained in Figure 2. a) 3 equiv 21, 6 equiv DIEA,
DMF, 14 h, RT, wash with DMF, CH
DIEA, DMF, 2 h, RT, wash with DMF, CH
with DMF, CH Cl ; e) 3 equiv 22 (activated with 2.7 equiv HBTU, 6 equiv DIEA, DMF, 15 min, RT), 2 h, RT, wash with DMF, CH
CH Cl Cl , DMF, DMF/DIEA, DMF; g) 9 equiv 23 (activated with 7 equiv HBTU, 6 equiv DIEA, DMF, 15 min, RT),
, 2 Â 30 s, 2 Â 10 min, wash with CH
4 h, 378C, wash with DMF, CH Cl ; h) 4 equiv 24 (activated with 3.8 equiv HOBt, 3.8 equiv DCC, DMF, 2.5 h, RT), 6 equiv DIEA, 2 h, RT, wash with DMF,
CH Cl ; i) 7 equiv 25 (activated with 4 equiv HBTU, 6 equiv DIEA, DMF, 20 min, RT), 14 h, 378C, wash with DMF, CH Cl , CH Cl /MeOH, CH Cl
j) saturated solution of MeNH
2
Cl
2
; b) 25% TFA in CH
Cl ; d) 3 equiv 12a ± d, 16 or 20 (activated with 2.7 equiv HBTU, 6 equiv DIEA, DMF, 15 min, RT), 2 h, RT, wash
Cl ; f) 50% TFA in
2
Cl
2
, 2 Â 30 s, 2 Â 10 min, wash with CH
2 2
Cl , DMF, DMF/DIEA, DMF; c) 3 equiv 21, 6 equiv
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
;
2
2 2
in MeOH, 1 h, 378C, wash with MeOH, CH Cl , MeOH.
À1
Table 1. Equilibrium association constants [m ] obtained in DNase I footprint- pairs A ¥ T, T ¥ A and C ¥ G with equal affinities. However, a G ¥
ing experiments with the EcoRI/PvuII restriction fragment of the plasmid
pDEH9 and the polyamides 1a ± d and 2 (see also Figure 5). All experiments
closer to the substituent on the U-turn as in a C ¥ G pair) still
were performed at least three times. Given are the average values and the
C base pair (where the exocyclic NH group of the G base is
2
causes a drop in affinity. Only U-pin 1 f with the unsubstituted
standard deviations (in parentheses).
turn tolerates all base pairs equally. Its affinity is again lower
Polyamide
5'-TGGTCA-3' 5'-TGGCCA-3' 5'-TGGGCA-3'
which was to be expected since this U-pin has one hydrogen
bond less than U-pins 1b, d and e and two hydrogen bonds less
than the hairpin polyamide 2. As a reference, six-ring hairpin
polyamides which have the same number of hydrogen bonds
(site ™A∫)
(site ™B∫)
(site ™C∫)
9
8
8
1
1
1
1
2
a (n ˆ 3, R ˆ CONHMe) 7.7  10 (1.1)
ꢀ 1 Â 10
ꢀ 2 Â 10
9
8
8
b (n ˆ 4, R ˆ CONHMe) 8.0  10 (1.2)
< 10
< 10
9
8
8
c (n ˆ 5, R ˆ CONHMe) 2.6  10 (0.5)
< 10
< 10
1
0
8
[a]
8 [a]
d (n ˆ 6, R ˆ CONHMe) 1.7  10 (0.3)
(5 Â 10 )
(5 Â 10 )
generally bind with an equilibrium association constant of K
a
1
0
8
8
(hairpin)
1.3 Â 10 (0.2)
ꢀ 4 Â 10
ꢀ 7 Â 10
8
À1 [18]
ꢀ
10 m .
[
a] At this threshold polyamide 1d binds nonspecifically to the DNA fragment.
Summary and Outlook
derived from the plasmid pDEH9 (cf. Figure 5 and Table 1).
Reducing the size of the ring substituent further increases the
tolerance for C or G adjacent to the turn residue: U-pin 1e
with the primary amide substituent, tolerates the three base
In summary, we have presented a new motif for minor groove-
binding pyrrole ± imidazole polyamides. The U-pins were
designed to have a G/C-tolerant turn. While U-pins might
Chem. Eur. J. 2003, 9, 3353 ± 3366
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3357

P. B. Dervan and A. Heckel
FULL PAPER
Figure 5. Results of DNase I footprinting experiments with the EcoRI/PvuII restriction fragment of the plasmid pDEH9 and the polyamides 1b, d and 2. In
the lower right corner the sequence of the restriction fragment is shown (insert used for cloning in bold-face) and the four binding sites of interest are labeled
A, B, C and D (for a discussion see text). The only match site for such a U-pin is site A. The numbers indicate the position on the DNA fragment.
3358
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2003, 9, 3353 ± 3366

U-Pin Polyamide Motif
3353±3366
spectrometer. The spectra were referenced as indicated, using reported
chemical shifts for the respective signals.[ Peaks were assigned by 2D
NMR spectroscopy and by comparison. UV/Vis spectra were recorded on a
Hewlett-Packard 8452A diode array spectrometer. High resolution mass
spectrometry was performed at the Mass Spectrometry Facility of the
University of California, Los Angeles (UCLA). All other mass spectra
were recorded at the mass spectrometry facility of the Division of
Chemistry and Chemical Engineering at Caltech. Analytical HPLC:
Beckman System Gold, 125 Solvent Module, 168 Detector, 508 Autosam-
pler, software: Gold Nouveau V1.7, unless otherwise noted the following
19]
conditions were used: column: Varian Microsorb MV 300-8 C18, flow:
À1
1
6
mLmin , solvents: A ˆ 0.1% TFA in H
2
O, B ˆ MeCN, gradient: 0 !
0% B linear in 30 min. Preparative HPLC: Beckman System Gold, 127P
Solvent Module, 166P Detector, columns: 1) Waters PrepLC 25 mm
module, 2) Hamilton PRP-1, unless otherwise noted the following con-
ditions were used: solvents: A ˆ 0.1% TFA in H
% B (0 min), 0% B (5 min), 20% B (30 min), 25% B (50 min), 35% B
60 min), 45% B (70 min), linear. Unless otherwise noted, enzymes were
2
O, B ˆ MeCN, gradient:
0
(
purchased from Roche. The oligonucleotides (inserts) used for the
preparation of the plasmids were obtained from the Oligonucleotide
Synthesis Facility of the Beckman Institute at the California Institute of
Technology. Sequencing of oligonucleotides was performed by the Se-
quence Analysis Facility, Division of Biology, California Institute of
Technology. Radioactively labeled nucleotides were purchased from
Perkin ± Elmer. The footprinting experiments were performed according
to published procedures[ using the general rule of e ˆ 8690 Lmol cm
20]
À1
À1
per ring in a polyamide for the quantification which turned out to also hold
for U-pins. To quantify the gels they were exposed over night on Kodak
storage phosphor screens (SO230, purchased from Molecular Dynamics).
To obtain the images, the storage phosphor screen was scanned on a
Typhoon 8600 phosphorimager from Molecular Dynamics. For the analysis
the software ImageQuant (version 5.1) was used and the equilibrium
affinity constants were determined according to previously published
methods.[ Abbreviations used: TMSE: trimethylsilylethyl, DCC: dicy-
clohexylcarbodiimide, HOBt: hydroxybenzotriazole, HBTU: 2-(1H-ben-
zotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa-fluorophosphate, DIEA:
H¸nig×s base, Phth: ortho-phthalimide.
Figure 6. Sequence of the EcoRI/PvuII restriction fragment of the plasmid
pAH2. The binding sites of interest are marked with boxes. The insert used
in the cloning process is shown in bold face.
20]
theoretically bind to DNA in different binding modes (Fig-
ure 4), only binding in the so-called ™normal∫ mode was
observed and the U-pins showed good selectivity for binding
to match over mismatch sites. U-pins tolerate several different
linker lengths with the exception of five linking carbon atoms.
Probing the influence of the substitution of the turn on G/C
tolerance, it was found that U-pin 1e is best for tolerating a
C ¥ G base pair. Finally, in order to target a DNA sequence in a
larger context which has a G ¥ C base pair in the turn region it
was necessary to remove all substituents on the U-turn, as
evident from the binding behavior of U-pin 1 f. The U-pin
motif extends the repertoire of DNA sites that can be targeted
by polyamides.
U-pins 1a ± f: The U-pins for this study were all prepared according to
[7]
reported solid phase synthesis protocols on oxime resin (Novabiochem,
À1
catalogue number 01-64-002, 0.48 mmolg ) and using the conditions
detailed in Scheme 3. The U-pins were stored at À808C and the purity of
this stockwas rechec ke d at the end of this study.
1
U-pin 1a: H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.97 (m, 2H;
‡
N(Py)CH
2
CH
2
CH
2
NH
3
), 2.18 (m, 2H; N(Py)CH
2
CH
2
CH
2
N(Im)), 2.66
‡
(
m, 8H; 2CONHCH
3
‡N(Py)CH
2
CH
2
CH
2
NH
3
), 3.75 (s, 3H; NMe), 3.76
(s, 3H; NMe), 3.82 (s, 3H; NMe), 3.83 (s, 3H; NMe), 3.99 (s, 3H; NMe),
‡
4.30 ± 4.32 (m, 4H; N(Py)CH
2
CH
2
CH
2
NH
3
‡N(Py)CH
2 2 2
CH CH N(Im)),
4
3
.46 (m, 2H; N(Py)CH
2
CH
2
CH
2
N(Im)), 6.79 ± 7.57 (m; arom. H), 7.64 (m,
‡
H; NH
3
), 7.93 ± 7.97 (m, 2H; 2CONHMe), 9.72 (s, 1H; amide NH), 9.89
(
(
1
s, 1H; amide NH), 9.96 (s, 1H; amide NH), 10.06 (s, 1H; amide NH), 10.42
s, 1H; amide NH), 10.50 (s, 1H; amide NH); analytical HPLC: t
R
ˆ
8.87 min; preparative HPLC: t
R
ˆ 46 min; UV/Vis (H
2
O): lmax ˆ 244,
3
17 nm; MS (MALDI): m/z: calcd for C50
58 20 8
H N O
: 1066.48; found:
‡
‡
‡
Experimental Section
1105.40 [M‡K] , 1089.43 [M‡Na] , 1067.44 [M‡H] .
1
U-pin 1b: H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.69 (m, 4H;
Compounds 21,[11a] 22,[11a] 24[2, 9] and 25[16] were obtained according to
literature procedures. All other chemicals used were purchased from
Aldrich. All reactions were performed under an Ar atmosphere and using
dry solvents. NMR spectra were recorded on a 300 MHz Varian Mercury
N(Py)CH
CH
N(Im)), 1.98 (m, 2H; N(Py)CH
CH
CH
NH
‡
),
2
CH
2
2
CH
2
2
2
2
3
‡
2.67 (m, 8H; 2CONHCH
3.82 (s, 3H; NMe), 3.83 (s, 3H; NMe), 4.00 (s, 6H; 2NMe), 4.29 (m, 2H;
N(Py)CH
3
‡N(Py)CH
2
CH
2
CH
2
NH
3
), 3.78 (s, 3H; NMe),
‡
2
CH
2
CH
2
CH
2
N(Im)), 4.35 (m, 2H; N(Py)CH
2
CH
2
CH
2
NH
3
),
À1
Table 2. Equilibrium association constants [m ] obtained in DNase I footprinting experiments with the EcoRI/PvuII restriction fragment of the plasmid
pAH2 and the polyamides 1b, d ± f and 2 (see also Figures 6 ± 8). All experiments were performed at least three times. Given are the average values and the
standard deviations (in brackets).
Polyamide
5'-TGGTCA-3'
5'-TGGTCT-3'
5'-TGGTCC-3'
5'-TGGTCG-3'
9
9
9
8
1
1
1
1
2
b (n ˆ 4, R ˆ CONHMe)
d (n ˆ 6, R ˆ CONHMe)
6.3 Â 10 (1.5)
8.9 Â 10 (1.4)
2.4 Â 10 (0.3)
1.8Â 10 (0.1)
9
9
9
8
4.1 Â 10 (0.9)
6.5 Â 10 (1.4)
2.0 Â 10 (1.0)
2.0Â 10 (0.5)
9
9
9
8
e (n ˆ 6, R ˆ CONH
f (n ˆ 6, R ˆ H)
(hairpin)
2
)
2.6 Â 10 (0.9)
2.6 Â 10 (1.0)
2.8 Â 10 (1.2)
4.1Â 10 (1.5)
8
8
8
8
2.3 Â 10 (0.4)
4.4 Â 10 (0.5)
2.9 Â 10 (0.8)
1.8Â 10 (0.2)
1
0
10
9
7
1.3 Â 10 (0.1)
1.4 Â 10 (0.1)
1.5 Â 10 (0.2)
< 5 Â 10
Chem. Eur. J. 2003, 9, 3353 ± 3366
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3359

P. B. Dervan and A. Heckel
FULL PAPER
Figure 7. Results of DNase I footprinting experiments with the EcoRI/PvuII restriction fragment of the plasmid pAH2 (see Figure 6) and the polyamides
1
d ± f and 2. The numbers indicate the base-pair position and correspond to those in Figure 6.
‡
‡
4
.44 (m, 2H; N(Py)CH
2
CH
2
CH
2
CH
2
N(Im)), 6.79 ± 7.57 (m; arom. H), 7.65
for C51
1081.51 [M‡H] .
H
60
N
20
O
8
‡
: 1080.49; found: 1119.50 [M‡K] , 1103.47 [M‡Na] ,
‡
(
m, 3H; NH
3
), 7.90 ± 7.95 (m, 2H; 2CONHMe), 9.70 (s, 1H; amide NH),
9
1
1
.88 (s, 1H; amide NH), 9.94 (s, 1H; amide NH), 10.02 (s, 1H; amide NH),
0.40 (s, 1H; amide NH), 10.44 (s, 1H; amide NH); analytical HPLC: t
9.58 min; preparative HPLC: t O): lmax (e) ˆ 250
ˆ 50 min; UV/Vis (H
1
R
ˆ
U-pin 1c: H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.21 (m, 2H;
R
2
N(Py)CH
2 2 2 2 2 2 2 2
CH CH CH CH N(Im)), 1.66 (m, 2H; N(Py)CH CH CH -
4
4
À1
À1
(
4.67 Â 10 ), 312 nm (6.91 Â 10 Lmol cm ); MS (MALDI): m/z: calcd
CH CH N(Im)), 1.75 (m, 2H; N(Py)CH CH CH CH CH N(Im)), 1.96
2
2
2
2
2
2
2
3360
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3353 ± 3366

U-Pin Polyamide Motif
3353±3366
CH
2
CH
2
CH
2
CH
2
CH
2
N(Im)), 3.82 (s, 3H; NMe), 3.83 (s, 3H; NMe), 3.99
‡
(
s, 6H; 2NMe), 4.34 (m, 2H; N(Py)CH
2
CH
2
CH
2
NH
3
), 4.42 (m, 2H;
N(Py)CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
N(Im)), 6.06 (m, 1H; arom. H), 6.56 (m,
‡
1
H; arom. H), 6.79 ± 7.58 (m; arom. H), 7.65 (m, 3H; NH
3
), 7.92 (m, H;
CONHMe), 9.72 (s, 1H; amide NH), 9.88 (s, 1H; amide NH), 9.90 (s, 1H;
amide NH), 9.95 (s, 1H; amide NH), 10.40 (s, 1H; amide NH), 10.47 (s, 1H;
amide NH); analytical HPLC: t
6 min, gradient: 0% B (0 min), 0% B (5 min), 25% B (60 min), 35% B
90 min), 45% B (100 min), linear; UV/Vis (H
R
ˆ 22.93 min; preparative HPLC: t ˆ
R
7
(
2
O): lmax (e) ˆ 250, 314 nm;
MS (MALDI): m/z: calcd for C51
H
61
N
19
O
7
: 1051.50; found: 1090.72
‡
‡
‡
[
M‡K] , 1074.72 [M‡Na] , 1052.80 [M‡H] .
Hairpin-polyamide 2: This compound was prepared according to reported
solid phase synthesis strategies[ on oxime resin (Novabiochem, catalogue
7]
À1
number 01-64-002, 0.48 mmolg ) and using the conditions detailed in
1
Scheme 3. H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.80 (m, 2H;
‡
NCH
2
CH
2
CH
2
CO (GABA)), 2.00 (m, 2H; N(Py)CH
2
CH
2
CH
2
NH
3
), 2.36
3
3
(
4
t, J(H,H) ˆ 6.6 Hz, 2H; NCH
2
CH
2
CH
2
CO (GABA)), 2.68 (d, J(H,H) ˆ
‡
.4 Hz, 3H; CONHCH
CH CH
3
), 2.76 (m, 2H; N(Py)CH
2
CH
2
CH
2
NH
3
), 3.20 (m,
Figure 8. Graphical representation of the binding affinities of the poly-
amides 1b, d ± f and 2 to the respective binding sites on the EcoRI/PvuII
restriction fragment of pAH2. For the numerical values see Table 2.
2H; NCH
CO (GABA)), 3.54 (s, 3H; NMe), 3.80 (s, 3H; NMe),
2
2
2
3.81 (s, 3H; NMe), 3.84 (s, 3H; NMe), 3.86 (s, 3H; NMe), 3.96 (s, 3H;
‡
NMe), 4.01 (s, 3H; NMe), 4.38 (m, 2H; N(Py)CH
2
CH
2
CH
2
NH
3
), 8.06 (m,
3
), 6.81 ±
3
7.67 (m; arom. H), 7.94 (d, J(H,H) ˆ 4.4 Hz, 1H; CONHCH
1
2 2 2
H; NHCH CH CH CO (GABA)), 9.74 (s, 1H; amide NH), 9.92 (s, 1H;
‡
(
m, 2H; N(Py)CH
2
CH
2
CH
2
NH
3
), 2.67 (m, 8H; 2CONHCH
3
‡
amide NH), 9.96 (s, 1H; amide NH), 10.00 (s, 1H; amide NH), 10.05 (s, 1H;
amide NH), 10.29 (s, 1H; amide NH), 10.45 (s, 1H; amide NH); analytical
‡
N(Py)CH
2
CH
2
CH
2
NH
3
), 3.78 (s, 3H; NMe), 3.82 (s, 3H; NMe), 3.83 (s,
3
H; NMe), 3.99 (s, 6H; 2NMe), 4.24 (m, 2H; N(Py)CH
2
CH
2
CH
2
CH
2
CH
2
-
HPLC: t ˆ 19.30 min (column: Varian Microsorb MV 100-5C18, 250 Â
R
‡
N(Im)), 4.35 (m, 2H; N(Py)CH
N(Py)CH
NH
amide NH), 9.95 (s, 1H; amide NH), 10.03 (s, 1H; amide NH), 10.42 (s, 1H;
amide NH), 10.46 (s, 1H; amide NH); analytical HPLC: t
preparative HPLC: t
2
CH
2
CH
2
NH
3
), 4.40 (m, 2H;
4.6 mm); preparative HPLC: t ˆ 49.5 min; UV/Vis (H O): l ˆ 236,
R
2
max
2
CH
2
CH
2
CH
2
CH
2
N(Im)), 6.78 ± 7.58 (m; arom. H), 7.66 (m, 3H;
52 61 21 9
314 nm; MS (MALDI): m/z: calcd for C H N O : 1123.50; found:
‡
‡
‡
‡
3
), 7.93 (m, 2H; 2CONHMe), 9.72 (s, 1H; amide NH), 9.89 (s, 1H;
1162.40 [M‡K] , 1146.39 [M‡Na] , 1124.41 [M‡H] .
,2,2-Trichloro-1-(4-nitro-1H-pyrrol-2-yl)-ethanone (4): 2,2,2-Trichloro-1-
1H-pyrrol-2-yl)-ethanone (209 g, 0.984 mol) was dissolved in Ac O (1 L)
(113 mL) was
2
(
R
ˆ 20.18 min;
2
R
ˆ 54 min; UV/Vis (H
2
O): lmax (e) ˆ 244, 311 nm; MS
and the solution was cooled to À158C. Then fuming HNO
3
‡
(
MALDI): m/z: calcd for C52
H
62
N
20
O
8
: 1094.51; found: 1138.48 [M‡K] ,
added very carefully, so that the temperature of the reaction mixture was
kept between À20 and À158C. After the addition was finished the
temperature of the reaction mixture was slowly allowed to come to RTand
stirring was continued over night. Then the mixture was poured into ice
water. The precipitate was collected and dried. After coevaporating with
toluene several times the residue was again dried and recrystallized from
‡
‡
1
117.51 [M‡Na] , 1095.55 [M‡H] .
1
U-pin 1d: H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.23 (m, 4H;
N(Py)CH
CH CH CH
2
CH
2 2 2 2 2 2 2
CH CH CH CH N(Im)), 1.61 (m, 2H; N(Py)CH CH -
2
2
2
CH
2
N(Im)), 1.73 (m, 2H; N(Py)CH
2
CH
2
CH
2
CH
2
CH
2
CH2-
‡
N(Im)), 1.97 (m, 2H; N(Py)CH
HCH
2
CH
2
CH
2
NH
3
), 2.67 (m, 8H; 2CON-
‡
3
‡N(Py)CH
2
CH
2
CH
2
NH
3
), 3.78 (s, 3H; NMe), 3.82 (s, 3H; NMe),
CHCl
3
/EtOH 95:5. Thus, compound 4 was isolated as beige solid (139 g,
3
.83 (s, 3H; NMe), 3.99 (s, 6H; 2NMe), 4.22 (m, 2H;
N(Py)CH CH CH CH CH CH N(Im)), 4.35 (m, 2H; N(Py)CH
CH CH NH ), 4.38 (m, 2H; N(Py)CH CH CH CH CH CH N(Im)),
.76 ± 7.58 (m; arom. H), 7.65 (m, 3H; NH ), 7.92 (m, 2H; 2CONHMe),
0
.540 mol, 55%). The spectroscopic data were in accord with literature
-
[2]
2
2
2
2
2
2
2
data.
‡
2
2
3
2
2
2
2
2
2
‡
4-Nitro-1H-pyrrole-2-carboxylic-2-trimethylsilanyl ethyl ester (5): NaH
0.8 g, 60%, 20.2 mmol, 0.2 equiv) was suspended in THF and 2-trime-
6
9
1
3
(
.72 (s, 1H; amide NH), 9.90 (s, 1H; amide NH), 9.96 (s, 1H; amide NH),
0.03 (s, 1H; amide NH), 10.43 (s, 1H; amide NH), 10.47 (s, 1H; amide
thylsilanyl-ethanol (16 mL, 13.2 g, 111 mmol, 1.1 equiv) was slowly added.
Then compound 4 (26 g, 101.0 mmol, 1 equiv) was dissolved in THF and
slowly added. After stirring at RT for 14 h the reaction mixture was
NH); analytical HPLC: t
gradient: 0% B (0 min), 0% B (5 min), 25% B (60 min), 35% B (90 min),
R
ˆ 22.28 min; preparative HPLC: t ˆ 73 min,
R
2 4 2 2
neutralized with conc. H SO and the solvent was evaporated. Then CH Cl
4
5% B (100 min), linear; UV/Vis (H
2
O): lmax (e) ˆ 252, 312 nm; MS
‡
(30 mL) and petroleum ether (80 mL) were added and the mixture was
kept in a freezer for several hours. After filtration, compound 5 was
isolated as a yellow powder (25.07 g, 97.8 mmol, 97%). The spectroscopic
(
MALDI): m/z: calcd for C53
H
64
N
20
‡
O
8
: 1108.53; found: 1147.45 [M‡K] ,
‡
1
131.49 [M‡Na] , 1109.53 [M‡H] .
1
U-pin 1e: H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.24 (m, 4H;
[2]
data were in accord with literature data.
N(Py)CH
CH CH CH
2
CH
2 2 2 2 2 2 2
CH CH CH CH N(Im)), 1.62 (m, 2H; N(Py)CH CH -
4
-tert-Butoxycarbonylamino-1H-pyrrole-2-carboxylic-2-trimethylsilanyl
2
2
2
CH
2
N(Im)), 1.73 (m, 2H; N(Py)CH
2
CH
2
CH
2
CH
2
CH
2
CH2-
‡
ethyl ester (6): In a 1 L bomb compound 5 (75 g, 293 mmol, 1.0 equiv) was
suspended in EtOAc (500 mL) and EtOH (150 mL) and the suspension was
purged with Ar. Then Pd/C (30 g, 10%) was added and the bomb was
N(Im)), 1.98 (m, 2H; N(Py)CH
HCH
2
CH
2
CH
2
NH
3
), 2.68 (m, 6H; CON-
‡
3
‡N(Py)CH
2
CH
2
CH
2
NH
3
), 3.79 (s, 3H; NMe), 3.83 (s, 3H;
NMe), 3.84 (s, 3H; NMe), 4.00 (s, 6H; 2NMe), 4.24 (m, 2H;
N(Py)CH CH CH CH CH CH N(Im)), 4.36 (m, 2H; N(Py)CH CH
), 4.42 (m, 2H; N(Py)CH CH CH CH CH CH N(Im)), 6.79 ±
), 7.93 (m, H; CONHMe), 9.73 (s,
pressurized with H
through Celite and most of the solvent was evaporated. Then an aqueous
solution of NaHCO and Boc O (96 g, 439.5 mmol, 1.5 equiv) were added
2
(25 bar). After 3 h the reaction mixture was filtered
2
2
2
2
2
2
2
2
-
‡
CH
2
NH
3
2
2
2
2
2
2
‡
7
1
1
.58 (m; arom. H), 7.66 (m, 3H; NH
3
3
2
and the mixture was stirred at RT for 1 h. After separation of the phases the
aqueous layer was extracted twice with EtOAc. The combined organic
H; amide NH), 9.90 (s, 1H; amide NH), 9.96 (s, 1H; amide NH), 10.01 (s,
H; amide NH), 10.43 (s, 1H; amide NH), 10.47 (s, 1H; amide NH);
phases were dried with MgSO
yellowish powder which was dissolved in CH
4
and the solvent was evaporated, affording a
Cl (100 mL), followed by
analytical HPLC: t
0
R
ˆ 21.15 min; preparative HPLC: t ˆ 70 min, gradient:
R
% B (0 min), 0% B (5 min), 25% B (60 min), 35% B (90 min), 45% B
2
2
addition of petroleum ether (800 mL). After keeping this mixture in a
freezer for several hours, filtration afforded compound 6 as a colorless
powder (80.3 g, 249 mmol, 84%). The spectroscopic data was in accord
with literature data.[
(
100 min), linear; UV/Vis (H
2
O): lmax (e) ˆ 252, 314 nm; MS (MALDI):
‡
m/z: calcd for C52
H
62
N
20
O
8
: 1094.51; found: 1117.25 [M‡Na] , 1095.24
‡
[
M‡H] .
2]
1
U-pin 1 f: H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.24 (m, 4H;
N(Py)CH CH CH CH CH CH N(Im)), 1.64 (m, 2H; N(Py)CH CH
CH CH CH CH
2
2
2
2
2
2
2
2
-
Compounds 7a ± c: Detailed example for the preparation of compound 7b:
NaH (20 g, 60%, 0.499 mol, 1.00 equiv) was suspended in THF (90 mL).
Then a solution of imidazole (34 g, 0.499 mol, 1.00 equiv) in THF (200 mL)
was slowly added. The suspension was heated to reflux and more THF
2
2
2
2
N(Im)), 1.72 (m, 2H; N(Py)CH
2
CH
2
CH
2
CH
2
CH
2
CH2-
‡
N(Im)), 1.97 (m, 2H; N(Py)CH
HCH
2
CH
2
CH
2
NH
3
), 2.67 (m, 6H; CON-
‡
3
‡N(Py)CH
2
CH
2
CH
2
NH
3
), 3.79 (m, 5H; NMe‡N(Py)CH
2
-
Chem. Eur. J. 2003, 9, 3353 ± 3366
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3361

P. B. Dervan and A. Heckel
FULL PAPER
(
0
80 mL) was added. After dissolving acetic 4-bromo-butyl ester (99.3 g,
.509 mol, 1.02 equiv) in THF (100 mL) this solution was added to the
reaction mixture. After heating to reflux for 20 h the reaction was allowed
to cool to room temperature, H O (1 mL) was added and the solvent was
evaporated. The residue was partitioned between CH Cl and H O and the
aqueous phase was extracted twice with CH Cl . The combined organic
layers were extracted with brine, dried with MgSO and the solvent was
and stirred over night. After quenching with H
evaporated and H O and CH Cl were added. The aqueous layer was
extracted twice with CH Cl . The combined organic layers were extracted
with brine. Then most of the solvent was evaporated and aqueous HCl (1m)
was added. After separation of the layers the organic phase was extracted
twice with dilute aqueous HCl. The combined aqueous layers were
2
O (1 mL), the solvent was
2
2
2
2
2
2
2
2
2
2
2
4
neutralized with K
2 3
CO and the product was extracted three times with
evaporated. Then the remaining orange oil was distilled under vacuum. To
remove traces of mineral oil the product was dissolved in dilute aqueous
HCl and the solution was extracted with petroleum ether. After neutral-
CH Cl . After drying with MgSO
compound 7d as slightly brownish oil (22.8 g, 108 mmol, 84%). H NMR
2
2
4
the solvent was evaporated, affording
1
(300 MHz, CDCl
3
, 258C, CHCl
3
): d ˆ 1.32 ± 1.38 (m, 4H; CH
2
), 1.62 (quint.,
3
3
ization with K
2
CO
3
the aqueous phase was extracted with CH
2
Cl
2
(three
J(H,H) ˆ 6.6 Hz, 2H; CH
2
), 1.79 (quint., J(H,H) ˆ 7.1 Hz, 2H; CH
2
), 2.04
3
3
times). The combined organic layers were dried with MgSO
4
and the
(s, 3H; OAc), 3.94 (t, J(H,H) ˆ 7.1 Hz, 2H; CH
2
), 4.04 (t, J(H,H) ˆ
solvent was evaporated to obtain spectroscopically pure compound 7b as
2
6.6 Hz, 2H; CH ), 6.91 (s, 1H; arom. H), 7.06 (s, 1H; arom. H), 7.46 (s,
1
13
colorless liquid (76.1 g, 0.418 mol, 84%). H NMR (300 MHz, CDCl
3
,
1H; arom. H); C NMR (75 MHz, CDCl
3
, 258C, CDCl
3
): d ˆ 21.01, 25.49,
2
58C, CHCl
3
): d ˆ 1.58 ± 1.67 (m, 2H; CH
2
), 1.81 ± 1.91 (m, 2H; CH
2
), 2.04
26.23, 28.42, 30.98, 46.87, 64.16, 118.58, 129.28, 136.87, 170.93; IR (CHCl
3
):
3
3
(
s, 3H; OAc), 3.99 (t, J(H,H) ˆ 7.1 Hz, 2H; CH
2
), 4.07 (t, J(H,H) ˆ
nÄ ˆ 2942 (s), 2863 (m), 1732 (s), 1509 (s), 1465 (m), 1390 (m), 1368 (s), 1251
À1
6
1
2
1
.6 Hz, 2H; CH
H; arom. H); 13C NMR (75 Hz, CDCl
7.78, 46.62, 63.45, 118.62, 129.20, 136.84, 170.82; IR (CHCl
2
), 6.92 (s, 1H; arom. H), 7.08 (s, 1H; arom. H), 7.58 (s,
(s), 1110 (m), 1078 (s), 910 cm (m); HR-MS (ESI): calcd for C11
H
18
N
2
O
2
:
‡
3
, 258C, CDCl
3
): d ˆ 21.01, 25.75,
210.1368; found: 211.1441 [M‡H] .
3
): nÄ ˆ 2966 (m),
Compounds 8a ± d: Detailed example for the preparation of compound 8b:
733 (s), 1508 (m), 1451 (w), 1390 (w), 1367 (m), 1224 (s), 1111 (w), 1078
Cl
After cooling the solution to 58C, compound 7b (30.6 g, 0.168 mol,
equiv), diluted with CH Cl (50 mL), was slowly added so that the
3
temperature did not exceed 108C. After stirring for 10 min at 58C, Et N
3 2 2
CCOCl (20.6 mL, 33.6 g, 0.185 mol, 1.1 equiv) was diluted with CH Cl .
À1
(
m), 1049 (m), 910 cm (w); HR-MS (ESI): calcd for C
9
H
14
N
2
O
2
: 182.1055;
‡
found: 183.113 [M‡H] .
1
2
2
Compound 7a: Obtained similarly; the acetic 3-bromo-propyl ester used
was obtained by diluting 3-bromo-propanol (50 g, 0.36 mol, 1 equiv) with
(23.4 mL, 0.168 mol, 1 equiv) was slowly added so that the temperature of
CH
.36 mol, 1 equiv) and Et
for 20 min an aqueous solution of NaHCO
separated. The organic layer was extracted once more with aqueous
NaHCO and then with brine. After drying with MgSO the solvent was
2
Cl
2
(300 mL) and adding a preformed mixture of Ac
N (50 mL, 0.36 mol, 1 equiv). After stirring at RT
was added and the phases were
2
O (34 mL,
the reaction mixture did not exceed 158C. After the addition was finished
0
3
the reaction mixture is diluted with CH
2 2
Cl and stirred at RT for 10 min.
Then the solvent was evaporated and EtOAc and an aqueous solution of
3
NaHCO
NaHCO
3
3
were added. The organic layer was again extracted with aqueous
and then with a 5% aqueous solution of citric acid. After drying
3
4
evaporated and the residue distilled under reduced pressure, affording
acetic 3-bromo-propyl ester as colorless oil (53.0 g, 0.292 mol, 81%). Then,
using imidazole (32.0 g, 0.470 mol, 1 equiv), acetic 3-bromo-propyl ester
4
the organic layer with MgSO , the solvent was evaporated, affording the
crude product as brownish oil (45.6 g, 0.139 mol, 83%). This product was
sufficiently pure for the following reactions. Since the product decomposes
very fast (even when kept at À208C) it was always immediately used for
further reactions. To obtain an spectroscopically pure sample, the crude
(
86.7 g, 0.479 mol, 1.02 equiv) and NaH (18.8 g, 60%, 0.470 mol, 1 equiv),
compound 7a was isolated as slightly yellowish oil (49.1 g, 0.292 mol, 62%).
1
R
f
(hexane/acetone 3:2) ˆ 0.38; H NMR (300 MHz, CDCl
3
, 258C, CHCl
CH CH
3
):
),
product was dissolved in CH
yellowish oil. R
2
Cl
2
and filtered through silica gel, affording a
3
1
d ˆ 2.03 (s, 3H; OAc), 2.07 (quint., J(H,H) ˆ 6.0 Hz, 2H; CH
2
2
2
f
(hexane/acetone 3:2) ˆ 0.56; H NMR (300 MHz, CDCl
3
,
3
4
.03 (m, 4H; CH
2
CH
2
CH
2
), 6.89 (d, J(H,H) ˆ 1.1 Hz, 1H; arom. H), 7.04
258C, TMS): d ˆ 1.72 (m, 2H; CH
2
), 1.90 (m, 2H; CH
2
), 2.07 (s, 3H; OAc),
),
d, 3J(H,H) ˆ 1.1 Hz, 1H; arom. H), 7.47 (s, 1H; arom. H); C NMR
13
3
3
(
(
4.12 (t, J(H,H) ˆ 6.0 Hz, 2H; CH
2
), 4.45 (t, J(H,H) ˆ 7.1 Hz, 2H; CH
2
1
3
75 MHz, CDCl
3
, 258C, CDCl
3
): d ˆ 20.84, 30.16, 43.68, 60.82, 118.60,
7.23 (s, 1H; arom. H), 7.38 (s, 1H; arom. H); C NMR (75 MHz, CDCl
258C, CDCl
): d ˆ 21.00, 25.67, 27.59, 49.20, 63.40, 127.49, 130.62, 135.35,
170.84, 171.88; IR (CHCl
3
,
1
(
8
29.45, 136.93, 170.52; IR (CHCl
w), 1392 (w), 1369 (m), 1225 (s), 1110 (m), 1079 (m), 1050 (m), 910 (m),
18 cm (m); HR-MS (EI): calcd for C
3
): nÄ ˆ 2968 (m), 1738 (s), 1508 (m), 1454
3
3
): nÄ ˆ 3002 (m), 2966 (m), 1732 (s), 1698 (m), 1662
À1
8
H
12
N
2
O
2
: 168.0899; found:
(s), 1583 (w), 1502 (m), 1466 (m), 1438 (w), 1387 (s), 1366 (s), 1394 (m),
‡
À1
1
68.0891 [M] .
1246 (s), 1170 (w), 1120 (w), 1050 (w), 916 cm (w).
Compound 8a: Obtained similarly, using compound 7a (10 g, 59 mmol,
Compound 7c: Obtained similarly, using imidazole (40.0 g, 0.587 mol,
equiv), acetic 5-bromo-pentyl ester (125.21 g, 0.599 mol, 1.02 equiv) and
NaH (23.5 g, 60%, 0.587 mol, 1 equiv), and isolated as colorless oil (77.5 g,
1
5
3 3
equiv), Cl CCOCl (7.2 mL, 11.8 g, 65 mmol, 1.1 equiv) and Et N (8.3 mL,
1
9 mmol, 1 equiv) and isolated as slightly yellowish liquid (11.8 g, 40 mmol,
1
1
67%). R (hexane/acetone 3:2) ˆ 0.52; H NMR (300 MHz, CDCl , 258C,
0
.395 mol, 67%). H NMR (300 MHz, CDCl
3
, 258C, CHCl
3
): d ˆ 1.25 ± 1.38
f
3
3
CHCl
3
): d ˆ 2.06 (s, 3H; OAc), 2.17 (quint., J(H,H) ˆ 6.6 Hz, 2H; CH
2
),
(
m, 2H; CH ), 1.58 ± 1.67 (m, 2H; CH ), 1.73 ± 1.84 (m, 2H; CH
2
2
2
), 2.01 (s,
3
3
3
3
4.11 (t, J(H,H) ˆ 6.0 Hz, 2H; CH ), 4.51 (t, J(H,H) ˆ 7.1 Hz, 2H; CH ),
3
H; OAc), 3.93 (dt, J(H,H) ˆ 1.6, 7.1 Hz, 2H; CH
2
), 4.02 (dt, J(H,H) ˆ
2
2
13
3
7.22 (s, 1H; arom. H), 7.37 (s, 1H; arom. H); C NMR (75 MHz, CDCl ,
1.6, 6.6 Hz, 2H; CH
2
), 6.88 (dd, J(H,H) ˆ 1.6, 2.7 Hz, 1H; arom. H), 7.03 (s,
3
1
3
258C, CDCl ): d ˆ 20.93, 29.76, 46.68, 60.86, 94.76, 127.71, 130.67, 135.42,
1
H; arom. H), 7.46 (s, 1H; arom. H); C NMR (75 MHz, CDCl
3
, 258C,
3
1
70.62, 171.90; IR (CHCl
3
): nÄ ˆ 2998 (m), 1738 (s), 1699 (s), 1468 (m), 1387
CDCl
3
): d ˆ 20.98, 23.03, 28.06, 30.68, 46.81, 63.87, 118.59, 129.20, 136.84,
À1
(
s), 1369 (m), 1242 (s), 1162 (w), 1078 (w), 1049 (m), 1018 (m), 918 cm (w).
1
70.85; IR (CHCl
3
): nÄ ˆ 2946 (m), 2867 (s), 1731 (s), 1509 (m), 1453 (w),
À1
1
391 (w), 1367 (m), 1245 (s), 1110 (m), 1078 (m), 1049 (m), 910 cm (w).
Compound 8c: Obtained similarly, using compound 7c (77.5 g, 0.395 mol,
1
0
9
3 3
equiv), Cl CCOCl (79.1 g, 0.435 mol, 1.1 equiv) and Et N (55 mL,
.395 mol, 1 equiv) and isolated as yellowish liquid (132.2 g, 0.387 mol,
Compound 7d: Triphenylphosphine (33.9 g, 129 mmol, 1 equiv) and
imidazole (8.8 g, 129 mmol, 1 equiv) were dissolved in CH Cl . After
cooling with an ice bath, I (32.8 g, 129 mmol, 1 equiv) was slowly added.
2
2
1
8%). R
f
(hexane/acetone 3:2) ˆ 0.57; H NMR (300 MHz, CDCl
3
, 258C,
), 1.88 (m, 2H; CH ), 2.05
), 4.44 (t, J(H,H) ˆ
2
CHCl
3
): d ˆ 1.45 (m, 2H; CH
2
), 1.71 (m, 2H; CH
2
2
After the addition was finished, the reaction mixture was stirred for
another 10 min at RT. Then acetic 6-hydroxy-hexyl ester (20.69 g,
3
3
(
s, 3H; OAc), 4.07 (t, J(H,H) ˆ 6.6 Hz, 2H; CH
2
1
3
[
12]
7.7 Hz, 2H; CH
75 MHz, CDCl
4.73, 127.44, 130.31, 135.04, 170.60, 171.49; IR (CHCl
2
), 7.27 (s, 1H; arom. H), 7.36 (s, 1H; arom. H); C NMR
1
29 mmol, 1 equiv)–prepared according to a literature procedure
–
(
9
3
, 258C, CDCl
3
): d ˆ 20.81, 22.80, 27.88, 30.33, 49.26, 63.68,
was diluted with CH Cl and slowly added to the previous reaction mixture,
2
2
): nÄ ˆ 2998 (m), 2958
which had again been cooled with an ice bath. After the addition was
finished the reaction mixture was stirred for another 10 min at RT and
3
(
m), 2866 (w), 1732 (s), 1699 (s), 1662 (m), 1468 (m), 1437 (w), 1387 (s),
À1
1
367 (m), 1250 (s), 1167 (w), 1068 (w), 1047 (w), 1020 (m), 918 cm (w).
extracted twice with an aqueous solution of Na
2
S
2
O
3
and then with brine.
The organic layer was then dried with MgSO
4
and the solvent was
Compound 8d: Obtained similarly, using compound 7d (1.87 g, 8.89 mmol,
1 equiv), Cl CCOCl (1.09 mL, 9.78 mmol, 1.1 equiv) and Et N (1.24 mL,
8.89 mmol, 1 equiv), and isolated as yellowish oil (3.16g, 8.89 mmol, 99%).
evaporated, affording a colorless solid (67.9 g) which was dissolved in THF.
Then NaH (5.0 g, 60%, 125 mmol, 0.97 equiv) was washed with hexane and
suspended in THF. After imidazole (8.4 g, 125 mmol, 0.97 equiv) had been
dissolved in THF and added to the NaH suspension, the slurry was heated
to reflux and the THF solution of the iodide was added. Heating (reflux)
was continued for 2 h. Then the reaction mixture was allowed to cool to RT
3
3
1
R
f
(hexane/acetone 3:2) ˆ 0.50; H NMR (300 MHz, CDCl
3
, 258C, CHCl
), 1.85 (m, 2H; CH ), 2.05 (s, 3H;
), 4.41 (t, J(H,H) ˆ 7.7 Hz, 2H;
3
):
d ˆ 1.41 (m, 4H; 2CH
2
), 1.65 (m, 2H; CH
2
2
3
3
OAc), 4.06 (t, J(H,H) ˆ 6.6 Hz, 2H; CH
2
1
3
2
CH ), 7.22 (s, 1H; arom. H), 7.37 (s, 1H; arom. H); C NMR (75 MHz,
3362
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3353 ± 3366

U-Pin Polyamide Motif
3353±3366
CDCl
3
, 258C, CDCl
3
): d ˆ 21.02, 25.49, 26.15, 28.43, 30.81, 49.57, 64.14,
were separated. The organic phase was extracted with brine, dried with
9
4.82, 127.46, 130.47, 135.28, 170.92, 171.76; IR (CHCl
3
): nÄ ˆ 2996 (m), 2942
MgSO
dissolved in MeCN, compound 6 (11.7 g, 36 mmol, 1.7 equiv) and K
(5.0 g, 36 mmol, 1.7 equiv) were added and the reaction mixture was heated
to 808C for 22 h. After filtration the solvent was evaporated and CH Cl
and an aqueous solution of NaHCO were added. The aqueous layer was
extracted twice with CH Cl . Then the combined organic layers were
extracted with brine and dried with MgSO . Evaporation of the solvent
afforded the crude product as a brown foam (22.7 g). Purification by flash
N) afforded com-
(hexane/acetone
4
and the solvent was evaporated. Then the orange residue was
(
(
s), 2863 (m), 1732 (s), 1698 (s), 1469 (s), 1438 (m), 1387 (s), 1368 (s), 1308
m), 1243 (s), 1020 (s), 975 cm (s); MS (ESI): m/z (%): 355.2 (85)
2
CO
3
À1
‡
[
M‡H] , 313.2 (100), 295.2 (25), 213.1 (85).
2
2
3
Compounds 9a ± d: Detailed example for the preparation of compound 9b:
A solution of NaOEt was prepared by adding Na (3.18 g, 0.138 mol,
2
2
4
1
equiv) to EtOH (200 mL). After all Na had reacted, a solution of freshly
prepared compound 8b (45.3 g, 0.138 mol, 1 equiv) in EtOH was added and
the reaction mixture was stirred over night at RT. Then the reaction
column chromatography (hexane/acetone 2:1‡1% Et
3
pound 10b as slightly red foam (4.9 g, 9.4 mmol, 45%). R
f
mixture was carefully neutralized with conc. H
evaporated. After adding EtOAc and a saturated solution of NaHCO
layers were separated and the aqueous layer was extracted twice with
EtOAc. The organic layers were combined, dried with MgSO and the
solvent was evaporated, affording compound 9b (24.8 g, 0.117 mol, 85%)
as an orange oil which could be used without any further purification. To
obtain an spectroscopically pure sample, the crude product was filtered
2
SO
4
and the solvent was
1
1
:1) ˆ 0.59; H NMR (300 MHz, DMSO, 258C, TMS): d ˆ 0.06 (s, 9H;
3
the
3
3
SiMe
3
2
3
), 1.05 (t, J(H,H) ˆ 8.2 Hz, 2H; CH
2
SiMe
3
), 1.42 (t, J(H,H) ˆ 7.1 Hz,
H; OCH
COOCH
2
CH
3
), 1.48 (s, 9; tBu), 1.78 (m, 4H; 2CH
2
3
), 4.25 ± 4.43 (m, 8H;
4
2
‡2NCH ), 6.26 (brs, 1H; NH), 6.60 (d, J(H,H) ˆ 2.2 Hz, 1H;
2
13
arom. H), 7.06 (s, 1H; arom. H), 7.26 (s, 2H; arom. H); C NMR (75 MHz,
DMSO, 258C, DMSO): d ˆ À1.33 (SiMe ), 14.11 (OCH CH ), 16.94
CH SiMe ), 27.88 (NCH CH CH CH N), 28.18 (C(CH ), 47.24 (NCH ),
47.45 (NCH ), 60.58 (COOCH ), 61.31 (COOCH ), 78.43 (C(CH ), 107.56,
118.11, 123.06 (2C), 126.24, 128.64, 135.21, 152.50, 158.40, 159.99; IR
3
2
3
(
2
3
2
2
2
2
3
)
3
2
through silica gel using hexane/acetone 1:4 as solvent, affording a yellowish
oil. R
1
2
2
2
3 3
)
f
(hexane/acetone 1:3) ˆ 0.47; H NMR (300 MHz, CDCl
3
, 258C,
3
TMS): d ˆ 1.42 (t, J(H,H) ˆ 7.1 Hz, 3H; OCH
2
CH
3
), 1.60 (quint.,
),
2
OH), 4.36 ± 4.46 (m,
3
3
(CDCl
3
): nÄ ˆ 3450 (w), 2983 (m), 1712 (s), 1587 (w), 1539 (w), 1472 (m),
J(H,H) ˆ 6.6 Hz, 2H; CH
2
3
), 1.91 (quint., J(H,H) ˆ 7.1 Hz, 2 H ; CH
2
À1
1422 (m), 1401 (m), 1369 (m), 1253 (s), 1160 (s), 1118 cm (m); HR-MS
3
4
.20 (brs, 1H; OH), 3.66 (t, J(H,H) ˆ 6.0 Hz; 2H, CH
‡
(
MALDI): calcd for C25
Compound 10a: Obtained similarly, using PPh
imidazole (2.7 g, 40 mmol, 1 equiv), I (10.2 g, 40 mmol, 1 equiv), com-
pound 9a (7.9 g, 40 mmol, 1 equiv), compound (23.5 g, 72 mmol,
H
40
N
4
O
6
Si: 520.2717; found: 543.2611 [M‡Na] .
H; OCH
2
CH
C NMR (75 MHz, CDCl
1.23, 61.41, 125.02, 129.00, 135.64, 158.67; IR (CHCl
3
‡NCH
2
), 7.11 (s, 1H; arom. H), 7.12 (s, 1H; arom. H);
1
3
, 258C, CDCl
3
): d ˆ 14.15, 27.68, 29.21, 48.11,
3
(10.5 g, 40 mmol, 1 equiv),
3
6
3
): nÄ ˆ 2984 (m), 1710
2
(
(
s), 1470 (m), 1422 (m), 1386 (m), 1258 (s), 1156 (w), 1118 (s); HR-MS
MALDI): calcd for C10
6
‡
H
16
N
2
O
3
: 212.1161; found: 235.1051 [M‡Na] ,
1.8 equiv) and K
red foam (5.1 g, 10 mmol, 25%). R
2
CO
3
(10.0 g, 72 mmol, 1.8 equiv) and isolated as lightly
‡
1
2
13.1232 [M‡H] .
f
(hexane/acetone 1:1) ˆ 0.40; H NMR
(
300 MHz, DMSO, 258C, DMSO): d ˆ 0.01 (s, 9H; SiMe
3
), 0.97 (t,
), 1.25 (t, J(H,H) ˆ 7.1 Hz, 3H;
), 1.41 (s, 9; tBu), 2.00 ± 2.10 (m, 2H; CH ), 4.16 ± 4.34 (m, 8H;
COOCH ), 6.62 (s, 1H; arom. H), 7.06 (s, 1H; arom. H), 7.12 (s,
‡2NCH
H; arom. H), 7.49 (s, 1H; arom. H), 9.14 (s, 1H; NH); C NMR (75 MHz,
), 14.88 (OCH CH ), 17.71
), 33.35 (NCH CH CH CH N), 46.19
), 61.32 (COOCH ), 62.13 (COOCH ), 79.22
), 108.46, 118.64, 119.06, 124.04, 126.75, 129.57, 136.14, 153.25,
59.11, 160.72; IR (CHCl
Compound 9a: Obtained similarly, using Na (400 mg, 17.5 mmol, 1 equiv)
and compound 8a (5.2 g, 17.5 mmol, 1 equiv) and isolated as yellow oil
3
3
J(H,H) ˆ 8.2 Hz, 2H; CH
2 3
SiMe
OCH
2
1
2
CH
3
2
1
(
2.5 g, 13 mmol, 72%). R
f
(hexane/acetone 1:3) ˆ 0.43; H NMR (300 MHz,
3
2
2
CDCl
3
, 258C, CHCl
3
): d ˆ 1.40 (t, J(H,H) ˆ 7.1 Hz, 3H; OCH
2 3
CH ), 2.03
1
3
quint., 3J(H,H) ˆ 6.0 Hz, 2H; CH
CH
), 3.61 (t, J(H,H) ˆ 6.0 Hz;
3
(
2
4
2
CH
2
2
DMSO, 258C, DMSO): d ˆ À0.57 (SiMe
3
2
3
3
H, CH
2
3
OH), 4.36 (quart.‡brs, J(H,H) ˆ 7.1 Hz, 3H; OCH
2
CH
), 7.11 (s, 1H; arom. H), 7.18 (s, 1H;
, 258C, CDCl
): d ˆ 13.96, 33.39,
4.91, 57.76, 61.16, 125.47, 128.69, 135.53, 158.51; IR (CDCl
): nÄ ˆ 3626 (w),
524 (w), 2964 (w), 1707 (s), 1473 (m), 1422 (s), 1386 (m), 1308 (m), 1257
3
‡OH),
(CH
(NCH
(C(CH
2
SiMe
3
), 28.95 (C(CH
3
)
3
2
2
2
2
.54 (t, J(H,H) ˆ 7.1 Hz, 2H; NCH
2
2
), 46.38 (NCH
2
2
2
1
3
arom. H); C NMR (75 MHz, CDCl
4
3
3
3
3 3
)
3
1
3
): nÄ ˆ 3449 (m), 3017 (m), 2982 (m), 1711 (s), 1567
(
m), 1538 (m), 1511 (w), 1470 (m), 1453 (m), 1423 (m), 1402 (m), 1369 (m),
(
9 14 2 3
s), 1182 (w), 1157 (w), 1116 (s); HR-MS (MALDI): calcd for C H N O :
À1
1
298 (w), 1252 (s), 1161 (s), 1117 (s), 1097 (s), 1062 (m), 861 (m), 840 cm
‡
‡
1
98.1004; found: 221.0895 [M‡Na] , 199.1076 [M‡H] .
(
m); HR-MS (ESI): calcd for C24
H
38
N
4
O
6
Si: 506.2561; found: 507.2630
Compound 9c: Obtained similarly, using Na (9.3 g, 0.404 mol, 1.05 equiv)
and compound 8c (132.2 g, 0.387 mmol, 1 equiv) and isolated as yellowish
oil (79.1 g, 0.35 mol, 90%). R
[M‡H]
‡
.
Compound 10c: Obtained similarly, using PPh
imidazole (2.7 g, 40 mmol, 1 equiv), I (10.15 g, 40 mmol, 1 equiv), com-
pound 9c (9.05 g, 40 mmol, 1 equiv), compound 6 (23.5 g, 72 mmol,
3
(10.5 g, 40 mmol, 1 equiv),
1
f
(hexane/acetone 1:3) ˆ 0.50; H NMR
2
(
(
300 MHz, CDCl
3
, 258C, TMS): d ˆ 1.43 (m, 5H; OCH
2 3 2
CH , CH ), 1.59
), 1.82 (quint., J(H,H) ˆ 7.7 Hz, 2H;
quint., 3J(H,H) ˆ 6.6 Hz, 2H; CH
3
2
1
.8 equiv) and K
2
CO
3
(10.0 g, 72 mmol, 1.8 equiv) and isolated as lightly
3
CH
2
), 3.11 (brs, 1H; OH), 3.62 (t, J(H,H) ˆ 6.6 Hz; 2H, CH
2
OH), 4.36 ±
), 7.10 (s, 1H; arom. H), 7.13 (s, 1H; arom.
H); C NMR (75 MHz, CDCl , 258C, CDCl
): d ˆ 14.10, 22.69, 30.84,
1.90, 48.27, 61.13, 61.79, 124.95, 128.94, 135.61, 158.59; IR (CHCl
): nÄ ˆ
986 (m), 2941 (m), 2867 (w), 1711 (s), 1473 (m), 1422 (s), 1386 (m), 1311
1
red foam (5.5 g, 10.3 mmol, 26%). R
f
(hexane/acetone 1:1) ˆ 0.55; H NMR
4
.43 (m, 4H; OCH
2
CH
3
‡NCH
2
(
300 MHz, DMSO, 258C, DMSO): d ˆ 0.02 (s, 9H; SiMe
3
), 0.98 (t,
1
3
3
3
3
3
J(H,H) ˆ 8.2 Hz, 2H; CH
.1 Hz, 3H; OCH CH
.32 (m, 8H; 2COOCH
arom. H), 7.11 (s, 1H; arom. H), 7.46 (s, 1H; arom. H), 9.10 (s, 1H; NH);
2
SiMe
3
), 1.17 (m, 2H; CH
2
), 1.26 (t, J(H,H) ˆ
3
2
3
7
4
2
3
), 1.42 (s, 9; tBu), 1.59 ± 1.70 (m, 4H; 2CH
2
), 4.15 ±
2
‡2NCH ), 6.59 (s, 1H; arom. H), 7.03 (s, 1H;
2
(
w), 1258 (s), 1156 (m), 1117 (m), 1068 (m); HR-MS (MALDI): calcd for
‡
‡
C
11
H
18
N
2
O
3
: 226.1317; found: 249.1208 [M‡Na] , 227.1386 [M‡H] .
1
3
C NMR (75 MHz, DMSO, 258C, DMSO): d ˆ À0.57 (SiMe
(OCH CH ), 17.71 (CH SiMe ), 23.69 (NCH CH CH CH CH N), 28.94
3
), 14.88
Compound 9d: Obtained similarly, using Na (202 mg, 8.8 mmol, 1.05 equiv)
2
3
2
3
2
2
2
2
2
and compound 8d (2.98 g, 8.38 mmol, 1 equiv) and isolated as yellowish oil
(C(CH ) ),
31.13
(NCH CH CH CH CH N),
31.44
3
3
2
2
2
2
2
1
(
(
1
6
1.75 g, 7.28 mmol, 87%).
300 MHz, CDCl
, 258C, TMS): d ˆ 1.25 ± 1.45 (m, 7H; OCH
.52 ± 1.60 (m, 2H; CH ), 1.71 ± 1.87 (m, 3H; CH
‡OH), 3.63 (t, J(H,H) ˆ
.0 Hz, 2H; CH OH), 4.37 ± 4.47 (m, 4H; OCH CH ), 7.15 (s, 1H;
‡NCH
arom. H), 7.29 (s, 1H; arom. H); C NMR (75 MHz, CDCl , 258C, CDCl ):
d ˆ 14.35, 25.31, 26.32, 31.23, 32.52, 48.42, 61.38, 62.58, 125.07, 129.29,
57.15; IR (CHCl
R
f
(hexane/acetone 1:1) ˆ 0.27;
H
NMR
(NCH CH CH CH CH N), 48.23 (NCH ), 48.54 (NCH ), 61.31
2
2
2
2
2
2
2
3
2
CH
3
3
‡2CH ),
2
2 2 3 3
(COOCH ), 62.04 (COOCH ), 79.18 (C(CH ) ), 108.24, 118.83, 123.76,
2
2
126.92, 129.35, 135.92, 153.26, 159.18, 160.76; IR (CHCl ): nÄ ˆ 3451 (m),
3
2
2
3
2
3020 (m), 2982 (m), 2957 (m), 1710 (s), 1586 (m), 1539 (m), 1508 (w), 1471
1
3
3
3
(m), 1456 (m), 1422 (m), 1403 (m), 1369 (m), 1252 (s), 1236 (s), 1161 (s),
1119 (m), 1088 (m), 1062 (m), 862 (m), 840 cm (m); HR-MS (ESI): calcd
À1
‡
1
3
): nÄ ˆ 3628 (m), 3392 (m), 2986 (s), 2940 (s), 2863 (s), 1711
for C H N O Si: 534.2874; found: 535.294 [M‡H] .
2
6
42
4
6
(
(
s), 1510 (w), 1473 (s), 1422 (s), 1386 (s), 1360 (m), 1311 (m), 1257 (s), 1156
s), 1117 (s), 1070 (s), 1052 (m), 1019 (m), 925 cm (m); HR-MS (MALDI):
Compound 10d: In a 1 L bomb compound 14 (9.36 g, 19.6 mmol, 1 equiv)
was dissolved in EtOAc (100 mL) and the solution was purged with Ar.
Then Pd/C (2.26 g, 10%) and Boc
added and the bomb was pressurized with H
À1
calcd for C12
H
20
N
2
O
3
: 240.1474; found: 241.1549 [M‡H]‡.
2
O (4.5 g, 20.6 mmol, 1.05 equiv) were
(35 bar). After stirring at RT
Compounds 10a ± c: Detailed procedure for the preparation of compound
2
1
1
0b: PPh
equiv) were dissolved in CH
3
(5.53 g, 21.1 mmol, 1 equiv) and imidazole (1.44 g, 21.1 mmol,
Cl and I (5.36 g, 21.1 mmol, 1 equiv) was
for 5 h all starting material had reacted (detection by NMR of a sample of
the reaction mixture since the starting material and the product are very
hard to separate by TLC). Then the reaction mixture was filtered through
Celite and the solvent was evaporated. Purification of the crude product by
2
2
2
added. After 10 min stirring at RT compound 9b (4.5 g, 21.1 mmol, 1 equiv)
was diluted with CH Cl and added. After 10 min stirring at RT the reaction
mixture was shaken with an aqueous solution of Na and the layers
2
2
2
S
2
O
3
flash column chromatography (hexane/acetone 2:1‡0.1% Et
3
N) afforded
Chem. Eur. J. 2003, 9, 3353 ± 3366
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3363

P. B. Dervan and A. Heckel
FULL PAPER
compound 10d (8.6 g, 15.7 mmol, 80%) as a colorless foam. R
f
(hexane/
88%). A part of it (5.49 g, 8.12 mmol, 1 equiv) was coupled with DCC
(7.71 mL, 1m in CH Cl ), HOBt (1.65 g, 12.18 mmol, 1.5 equiv), MeNH
2 2 2
(4.87 mL, 2m in THF, 9.74 mmol, 1.2 equiv) and DIEA (2.83 mL,
16.24 mmol, 2 equiv), affording compound 11c (3.30 g, 7.37 mmol, 91%)
1
acetone 3:2) ˆ 0.50; H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 0.01
3
(
2
1
(
s, 9H; SiMe
CH
.65 (m, 4H; 2NCH
3
), 0.98 (t, J(H,H) ˆ 7.7 Hz, 2H; CH
2
SiMe
3
), 1.20 (m, 4H;
3
2
), 1.26 (t, J(H,H) ˆ 7.1 Hz, 3H; OCH
2
CH
3
), 1.41 (s, 9H; tBu), 1.44 ±
‡2NCH ), 6.59
s, 1H; arom. H), 7.04 (s, 1H; arom. H), 7.10 (s, 1H; arom. H), 7.47 (s, 1H;
1
2
CH
2
), 4.14 ± 4.31 (m, 8H; 2COOCH
2
2
as yellowish foam. R
f
(hexane/acetone 2:3) ˆ 0.40; H NMR (300 MHz,
DMSO, 258C, DMSO): d ˆ 1.14 (m, 2H; NCH
2 2 2
CH CH ), 1.26 (t,
CH ), 1.42 (s, 9H; tBu), 1.56 ± 1.69 (m, 4H;
2 3
1
3
3
arom. H), 9.11 (s, 1H; NH); C NMR (75 MHz, DMSO, 258C, DMSO):
d ˆ À1.32 (SiMe ), 14.16 (OCH CH ), 16.97 (CH SiMe ), 25.56 (2 middle
CH ), 28.19 (C(CH ), 30.72 (NCH CH ), 31.10 (NCH CH ), 47.60
NCH ), 47.91 (NCH ), 60.55 (COOCH ), 61.27 (COOCH ), 78.42
C(CH ), 107.47, 118.10, 122.99, 126.18, 128.63, 135.21, 152.51, 158.46,
60.01; IR (CHCl
): nÄ ˆ 3450 (m), 3007 (m), 2983 (m), 2957 (m), 2863 (w),
710 (s), 1585 (m), 1537 (m), 1511 (w), 1470 (m), 1455 (m), 1422 (m), 1404
J(H,H) ˆ 7.1 Hz, 3H; OCH
3
3
2
3
2
3
2NCH
2
CH
2
), 2.63 (d, J(H,H) ˆ 4.4 Hz, 3H; CONHCH
3
), 4.16 ± 4.31 (m,
2
3
)
3
2
2
2
2
6H; COOCH
2
‡2NCH
2
), 6.54 (s, 1H; arom. H), 6.86 (s, 1H; arom. H), 7.04
3
(
(
2
2
2
2
(s, 1H; arom. H), 7.47 (s, 1H; arom. H), 7.85 (d, J(H,H) ˆ 4.4 Hz, 1H;
1
3
3
)
3
CONHMe), 9.03 (s, 1H; NH (Boc)); C NMR (75 MHz, DMSO, 258C,
CH ), 23.78 (NCH CH CH ), 26.36
), 31.18 (NCH CH ), 31.69 (NCH CH ),
), 61.33 (COOCH ), 78.90 (C(CH ), 103.92,
):
1
1
3
DMSO): d ˆ 14.91 (OCH
2
3
2
2
2
(COONHCH
48.17 (NCH
3
), 29.00 (C(CH
), 48.28 (NCH
3
)
3
2
2
2
2
(
(
5
m), 1252 (s), 1160 (s), 1119 (m), 1102 (m), 1084 (m), 1061 (m), 997 (w), 937
2
2
2
3 3
)
À1
w), 861 cm (m); HR-MS (ESI): calcd for C27
H
44
N
4
O
6
Si: 548.3030; found:
115.82, 122.93, 126.97, 129.35, 135.91, 153.36, 159.19, 162.14; IR (CHCl
3
49.3106 [M‡H]‡.
nÄ ˆ 3451 (w), 3007 (m), 2936 (m), 1713 (s), 1656 (m), 1591 (w), 1534 (s),
1
472 (m), 1422 (m), 1394 (m), 1369 (m), 1242 (s), 1161 (s), 1115 (m), 1094
Compounds 11a ± d: Detailed procedure for the preparation of compound
À1
(
m), 1069 (m), 997 cm (w); HR-MS (ESI): calcd for C22
H
33
N
5
O
5
:
1
1b: Compound 10b (4.74 g, 9.1 mmol, 1 equiv) was dissolved in THF and
4
47.2482; found: 448.2551 [M‡H]‡.
the solution was cooled in an ice bath. Then TBAF (27.3 mL, 1m solution in
THF, 27.3 mmol, 3 equiv) was added and the reaction mixture was stirred
Compound 11d: Obtained similarly, using compound 10d (8.6 g,
5.7 mmol, 1 equiv) and TBAF (47.1 mL, 1m in THF, 47.1 mmol, 3 equiv),
affording the tetrabutylammonium salt of the deprotected acid (9.25 g,
3.4 mmol, 85%). A part of it (2.00 g, 2.9 mmol, 1 equiv) was coupled with
DCC (2.75 mL, 1m in CH Cl , 2.75 mmol, 0.95 equiv), HOBt (588 mg,
.35 mmol, 1.5 equiv), MeNH (1.74 mL, 2m in THF, 3.48 mmol, 1.2 equiv)
and DIEA (1 mL, 5.80 mmol, 2 equiv), affording compound 11d (1.17 g,
for 14 h. Then the solvent was evaporated and CH
organic phase was extracted with aqueous citric acid (0.5m). The aqueous
phase was extracted twice with CH Cl . Then the organic phases were
combined and extracted with brine, dried with MgSO and the solvent was
evaporated, affording the tetrabutylammonium salt of the deprotected acid
5.4 g, 8.16 mmol, 90%) as a beige foam. An aliquot of this salt (1.0 g,
.5 mmol, 1 equiv) was dissolved in DMF and HOBt (306 mg, 2.3 mmol,
.5 equiv) and DCC (1.4 mL, 1m in CH Cl , 1.4 mmol, 0.95 equiv) were
added. After stirring at RT for 4 h the reaction mixture was filtered and
MeNH (0.8 mL, 2m in THF, 1.6 mmol, 1.05 equiv) and DIEA (261 mL,
.5 mmol, 1 equiv) were added. After stirring at RT for 20 min the solvent
was evaporated. EtOAc and an aqueous solution of NaHCO were added.
2 2
Cl was added. This
1
2
2
1
4
2
2
4
2
(
1
2
H
2
.53 mmol, 87%) as yellowish foam. R
f
(hexane/acetone 2:3) ˆ 0.50;
1
2
2
1
NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.17 (m, 4H;
), 1.26 (t, 3J(H,H) ˆ 7.1 Hz, 3H; OCH
NCH
2
CH
2
CH
2
2 3
CH ), 1.40 (s, 9H;
2
3
tBu), 1.43 ± 1.66 (m, 4H; 2NCH
CONHCH
H), 6.83 (s, 1H; arom. H), 7.02 (s, 1H; arom. H), 7.46 (s, 1H; arom. H), 7.83
2
CH
2
), 2.61 (d, J(H,H) ˆ 4.4 Hz, 3H;
1
3
), 4.13 ± 4.30 (m, 6H; COOCH
2
‡2NCH
2
), 6.51 (s, 1H; arom.
3
After separation of the layers, the aqueous phase was extracted twice with
EtOAc. The combined organic phases were then extracted with brine, dried
3
13
(
(
(
(
(
d, J(H,H) ˆ 4.9 Hz, 1H; CONHMe), 9.02 (s, 1H; NH (Boc)); C NMR
75 MHz, DMSO, 258C, DMSO): d ˆ 14.16 (OCH CH ), 24.51
COONHCH ), 25.58 (2NCH CH CH ), 28.23 (C(CH ), 30.27
NCH CH ), 31.32 (NCH CH ), 47.49 (NCH ), 47.61 (NCH ), 60.57
COOCH ), 78.15 (C(CH ), 103.13, 114.99, 122.12, 122.30, 126.18,
28.62, 135.20, 152.04, 152.61, 158.45, 161.39; IR (CHCl
2
3
with MgSO
raphy (hexane/acetone 2:3‡0.1% Et
4
and the solvent was evaporated. Flash column chromatog-
N) afforded compound 11b (473 mg,
3
2
2
2
3 3
)
3
2
2
2
2
2
2
0
.92 mmol, 61%) as colorless foam.
R
f
(hexane/acetone 2:3) ˆ 0.36;
2
3 3
)
1
3
H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.26 (t, J(H,H) ˆ 7.1 Hz,
1
3
3
): nÄ ˆ 3452 (w),
3
H; OCH
2
CH
3
), 1.41 (s, 9H; tBu), 1.57 (m, 4H; 2NCH
3H; CONHCH ), 4.21 ± 4.32
2
CH
(m,
2
), 2.62 (d,
6H;
006 (m), 2938 (m), 1714 (s), 1654 (m), 1591 (m), 1534 (m), 1472 (m), 1421
3
J(H,H) ˆ 4.4 Hz,
3
À1
(
m), 1255 (m), 1160 (m), 1115 (w), 1069 cm (w); HR-MS (ESI): calcd for
COOCH
2
‡2NCH
2
), 6.54 (s, 1H; arom. H), 6.86 (s, 1H; arom. H), 7.03 (s,
: 461.2638; found: 462.2714 [M‡H]‡.
23 35 5 5
C H N O
3
1
H; arom. H), 7.48 (s, 1H; arom. H), 7.86 (d, J(H,H) ˆ 4.9 Hz, 1H;
1
3
CONHMe), 9.04 (s, 1H; NH (Boc)); C NMR (75 MHz, DMSO, 258C,
DMSO): d ˆ 14.88 (OCH CH ), 25.28 (COONHCH ), 26.36 (NCH CH ),
8.99 (C(CH ), 29.17 (NCH CH ), 47.78 (NCH ), 48.04 (NCH ), 61.36
COOCH ), 78.94 (C(CH ), 103.99, 115.86, 122.98, 127.05, 129.39, 135.96,
Compounds 12a ± d: Detailed procedure for the preparation of compound
12b: Compound 11b (764 mg, 1.76 mmol, 1 equiv) was dissolved in a
2
3
3
2
2
2
(
3
)
3
2
2
2
2
solution of NaOH in MeOH/H O 1:1 (17.6 mL, 0.1m, 1.76 mmol, 1 equiv).
2
2
3
)
3
After shaking at 378C for 2 h the reaction mixture was lyophilized,
1
53.36, 159.17, 162.11; HR-MS (MALDI): calcd for C21
H
31
N
5
O
5
: 433.2325;
affording compound 12b as a colorless foam (712 mg, 1.67 mmol, 95%).
The Na salt of the product is remarkably stable (no decomposition
detectable by NMR or MS(ESI) after storage for seven months at À208C).
However, it was always freshly prepared before the solid phase synthesis.
‡
found: 456.2210 [M‡Na] .
Compound 11a: Obtained similarly, using compound 10a (5.0 g, 9.9 mmol,
equiv) and TBAF (29.6 mL, 1m in THF, 29.6 mmol, 3 equiv), affording
the tetrabutylammonium salt of the deprotected acid (5.0 g, 7.7 mmol,
8%). A part of it (4.92 g, 7.59 mmol, 1 equiv) was coupled with DCC
7.21 mL, 1m in CH Cl ), HOBt (1.54 g, 11.39 mmol, 1.5 equiv), MeNH
4.55 mL, 2m in THF, 9.11 mmol, 1.2 equiv) and DIEA (2.64 mL,
1
1
Upon acidification the product decarboxylates spontaneously. H NMR
(
2
300 MHz, DMSO, 258C, DMSO): d ˆ 1.41 (s, 9H; tBu), 1.56 (m, 4H;
7
(
(
3
NCH
2
CH
2
), 2.63 (d, J(H,H) ˆ 4.4 Hz, 3H; CONHCH
3
), 4.21 (brs, 2H;
2
2
2
NCH
6
2
), 4.43 (brs, 2H; NCH
.85 (s, 1H; arom. H), 7.07 (s, 1H; arom. H), 7.90 (d, J(H,H) ˆ 4.4 Hz, 1H;
2
), 6.57 (s, 1H; arom. H), 6.73 (s, 1H; arom. H),
3
1
5.18 mmol, 2 equiv), affording compound 11a (2.30 g, 5.5 mmol, 72%)
1
3
1
CONHMe), 9.05 (s, 1H; NH (Boc)); C NMR (75 MHz, DMSO, 258C,
DMSO): d ˆ 26.36 (COONHCH ), 29.00 (C(CH ), 34.15 (NCH CH ),
47.08 (NCH ), 47.97 (NCH ), 78.91 (C(CH ), 104.08, 115.91, 122.57, 122.98,
26.27, 145.69, 153.39, 162.14, 162.78; HR-MS (ESI): calcd for C19
as yellowish foam. R
f
(hexane/acetone 2:3) ˆ 0.20; H NMR (300 MHz,
3
3
3
)
3
2
2
DMSO, 258C, DMSO): d ˆ 1.25 (t, J(H,H) ˆ 7.1 Hz, 3H; OCH
2 3
CH ), 1.41
3
2
2
3 3
)
(
s, 9H; tBu), 2.09 (m, 2H; NCH
2
CH
2
CH
2
), 2.63 (d, J(H,H) ˆ 3.8 Hz, 3H;
1
4
27 5 5
H N O :
CONHCH
3
), 4.21 ± 4.32 (m, 6H; COOCH
2
‡2NCH
2
), 6.57 (s, 1H; arom.
À
05.2012; found: 404.1942 [MÀH] .
H), 6.86 (s, 1H; arom. H), 7.06 (s, 1H; arom. H), 7.49 (s, 1H; arom. H), 7.86
3
13
(
(
(
(
1
2
d, J(H,H) ˆ 4.4 Hz, 1H; CONHMe), 9.08 (s, 1H; NH (Boc)); C NMR
Compound 12a: Obtained similarly, using compound 11a (630 mg,
.5 mmol, 1 equiv) and a solution of NaOH in MeOH/H O 1:1 (15 mL,
75 MHz, DMSO, 258C, DMSO): d ˆ 14.89 (OCH CH ), 26.36
COONHCH ), 28.99 (C(CH ), 33.65 (NCH CH ), 46.00 (NCH ), 46.34
NCH ), 61.35 (COOCH ), 78.97 (C(CH ), 104.06, 115.60, 123.15, 123.25,
26.70, 129.55, 136.14, 153.36, 159.10, 162.05; IR (CHCl
2
3
1
2
3
3
)
3
2
2
2
0
9
2
.1m, 1.5 mmol, 1 equiv) and isolated as colorless foam (615 mg, 1.49 mmol,
2
2
3
)
3
1
9%). H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.41 (s, 9H; tBu),
3
): nÄ ˆ 3449 (m),
3
.05 (m, 2H; NCH
2
CH
2
), 2.63 (d, J(H,H) ˆ 3.3 Hz, 3H; CONHCH
3
), 4.17
),
936 (m), 1710 (s), 1656 (s), 1592 (m), 1547 (w), 1462 (m), 1384 (m), 1368
3
3
(
6
t, J(H,H) ˆ 7.1 Hz, 2H; NCH
2
), 4.42 (t, J(H,H) ˆ 7.1 Hz, 2H; NCH
2
À1
(
m), 1310 (w), 1156 (s), 1114 (s), 996 cm (w); HR-MS (ESI): calcd for
.58 (s, 1H; arom. H), 6.76 (s, 1H; arom. H), 6.82 (s, 1H; arom. H), 7.06 (s,
C
20
H
29
N
5
O
5
: 419.2169; found: 420.2232 [M‡H]‡.
3
1
H; arom. H), 7.92 (d, J(H,H) ˆ 3.8 Hz, 1H; CONHMe), 9.07 (s, 1H; NH
1
3
Compound 11c: Obtained similarly, using compound 10c (5.2 g, 9.7 mmol,
equiv) and TBAF (29.2 mL, 1m in THF, 29.2 mmol, 3 equiv), affording
the tetrabutylammonium salt of the deprotected acid (5.8 g, 8.6 mmol,
(Boc));
(COONHCH
C
NMR (75 MHz, DMSO, 258C, DMSO): d ˆ 26.40
1
3
), 29.01 (C(CH
3 3 2 2 2
) ), 34.33 (NCH CH ), 45.49 (NCH ), 46.35
(NCH ), 78.96 (C(CH )
2
3 3
), 104.10, 115.51, 122.36, 123.19, 126.51, 145.78,
3364
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3353 ± 3366

U-Pin Polyamide Motif
3353±3366
1
53.39, 162.12, 162.78; HR-MS (ESI): calcd for C18
H
25
N
5
O
5
: 391.1856;
tBu), 1.43 ± 1.66 (m, 4H; 2NCH
COOCH
‡2NCH
s, 1H; arom. H), 7.02 (s, 1H; arom. H), 7.38 (brs, 1H; CONH
2
CH
2
), 4.14 ± 4.30 (m, 6H;
‡
‡
found: 414.1735 [M‡Na] , 392.1916 [M‡H] .
Compound 12c: Obtained similarly, using compound 11c (671 mg,
.5 mmol, 1 equiv) and a solution of NaOH in MeOH/H O 1:1 (15 mL,
2
2
), 6.58 (s, 1H; arom. H), 6.70 (brs, 1H; CONH
), 7.46 (s, 1H;
arom. H), 8.99 (s, 1H; NH (Boc)); C NMR (75 MHz, DMSO, 258C,
DMSO): d ˆ 14.17 (OCH CH ), 25.54 (2 NCH CH CH ), 28.24 (C(CH ),
30.71 (NCH CH ), 31.26 (NCH CH ), 47.52 (NCH ), 47.61 (NCH ), 60.58
COOCH ), 78.13 (C(CH ), 104.40, 115.67, 121.90, 122.00, 128.64, 135.20,
152.67, 158.45, 162.71; IR (CHCl
): nÄ ˆ 3534 (w), 3449 (w), 3417 (w), 2984
m), 2938 (m), 2862 (m), 1713 (s), 1665 (s), 1599 (w), 1575 (m), 1536 (w),
2
), 6.84
(
2
1
3
1
0
9
2
2
3
2
2
2
3 3
)
.1m, 1.5 mmol, 1 equiv) and isolated as colorless foam (666 mg, 1.50 mmol,
1
2
2
2
2
2
2
9%). H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.11 (m, 2H; CH
2
),
(
2
3 3
)
1.42 (s, 9H; tBu), 1.62 (m, 4H; 2NCH
2
CH
2
), 2.63 (s, 3H; CONHCH
3
), 4.16
),
.55 (s, 1H; arom. H), 6.73 (s, 1H; arom. H), 6.85 (s, 1H; arom. H), 7.05 (s,
3
3
3
(
6
1
t, J(H,H) ˆ 7.1 Hz, 2H; NCH
2
), 4.40 (t, J(H,H) ˆ 7.1 Hz, 2H; NCH
2
(
1
3
1510 (w), 1467 (w), 1423 (w), 1323 (w), 1255 (m), 1160 (s), 1121 (w), 1070
H; arom. H), 7.89 (brs, 1H; NH); C NMR (75 MHz, DMSO, 258C,
), 26.38 (COONHCH ), 29.02 (C(CH ), 31.68
), 31.92 (NCH CH ), 47.51 (NCH ), 48.26 (NCH ), 78.88
), 104.05, 115.87, 122.62, 123.03, 126.24, 145.59, 153.43, 162.20,
62.90; HR-MS (ESI): calcd for C20 : 419.2169; found: 442.2051
À1
(
4
w), 998 cm (w); HR-MS (ESI): calcd for C22
H
33
N
5
O
5
: 447.2482; found:
DMSO): d ˆ 23.99 (CH
2
3
3 3
)
48.2558 [M‡H]‡.
(
NCH
2
CH
2
2
2
2
2
(
1
C(CH )
3 3
Compound 16: Obtained analogously to compound 12d, using compound
5 (31 mg, 69.3 mmol, 1 equiv) and a solution of NaOH in MeOH/H O 1:1
693 mL, 0.1m, 69.3 mmol, 1 equiv) (additional MeOH (1.5 mL) had to be
29 5 5
H N O
1
(
2
‡
‡
[
M‡Na] , 420.2231 [M‡H] .
Compound 12d: Obtained similarly, using compound 11d (27 mg,
added to completely dissolve the starting material) and isolated as
5
0
8.5 mmol, 1 equiv) and a solution of NaOH in MeOH/H
2
O 1:1 (585 mL,
1
yellowish foam (23 mg, 49.6 mmol, 72%). H NMR (300 MHz, DMSO,
.1m, 58.5 mmol, 1 equiv) (additional H O (1 mL) and MeOH (2 mL) had
2
258C, DMSO): d ˆ 1.15 (m, 4H; 2NCH CH CH ), 1.41 (s, 9H; tBu), 1.54 ±
2
2
2
to be added to completely dissolve the starting material) and isolated as
yellowish foam (22 mg, 45.7 mmol, 79%). H NMR (300 MHz, DMSO,
2
3
1
.62 (m, 4H; 2NCH
2
CH
2
), 4.16 (t, J(H,H) ˆ 6.6 Hz, 2H; NCH
2
), 4.40 (t,
1
3
J(H,H) ˆ 7.1 Hz, 2H; NCH
2
), 6.60 (s, 1H; arom. H), 6.72 (s, 1H; arom. H),
.84 (s, 1H; arom. H), 7.05 (s, 1H; arom. H); C NMR (75 MHz, DMSO,
58C, DMSO): d ˆ 1.15 (m, 4H; 2NCH
2
CH
2
CH
2
), 1.42 (s, 9H; tBu), 1.54 ±
13
6
3
1.62 (m, 4H; 2NCH
2
CH
2
), 2.63 (d, J(H,H) ˆ 4.4 Hz, 3H; CONHCH
3
),
258C, DMSO): d ˆ 25.75 (NCH CH CH ), 28.28 (C(CH ) ), 31.20
2
2
2
3 3
.15 (t, 3J(H,H) ˆ 6.6 Hz, 2H; NCH
), 4.40 (t, J(H,H) ˆ 6.6 Hz, 2H;
3
4
2
(
NCH
2
2 2 2 2 2
CH ), 31.39 (NCH CH ), 46.77 (NCH ), 47.57 (NCH ), 78.00
NCH
2
), 6.54 (s, 1H; arom. H), 6.72 (s, 1H; arom. H), 6.84 (s, 1H; arom. H),
(
C(CH
3
)
3
), 104.53, 115.77, 121.87, 122.32, 125.49, 144.94, 152.83, 162.82;
3
7
1
.04 (s, 1H; arom. H), 7.89 (d, J(H,H) ˆ 3.8 Hz, 1H; COONHMe), 9.04 (s,
‡
HR-MS (ESI): calcd for C20
H
29
N
5
O
5
: 419.2169; found: 442.2057 [M‡Na] .
H; NH (Boc)); 13C NMR (75 MHz, DMSO, 258C, DMSO): d ˆ 24.60
Compound 17: PPh
3
(5.25 g, 20 mmol, 1 equiv) and imidazole (1.36 g,
0 mmol, 1 equiv) were dissolved in CH Cl and I (5.08 g, 20 mmol,
equiv) was added. After stirring at RT for 10 min compound 9d (4.81 g,
0 mmol, 1 equiv) was dissolved in CH Cl and added. After stirring at RT
(COONHCH
(C(CH
3 3
(NCH ), 78.15 (C(CH )
3
), 25.63 (NCH
CH
), 103.24, 115.01, 121.71, 122.13, 122.38, 125.46,
2
CH
2
CH
2
), 25.78 (NCH
2
CH
2
CH
2
), 28.26
2
1
2
2
2
2
3
)
3
), 31.25 (NCH
2
2
), 31.44 (NCH
2
CH
2
), 46.76 (NCH
2
), 47.56
2
1
4
45.02, 152.63, 161.46, 162.07; HR-MS (ESI): calcd for C21
H
31
N
5
O
5
:
2
2
À
for 10 min the reaction mixture was shaken with an aqueous solution of
Na and the layers were separated. The aqueous layer was extracted
three times with CH Cl . Then the combined organic layers were extracted
with brine, dried with MgSO and the solvent was evaporated. The orange
33.2325; found: 432.2263 [MÀH] .
2 2 3
S O
Compound 14: PPh
3
(8.73 g, 33.3 mmol, 1 equiv) and imidazole (2.27 g,
Cl and I (8.45 g, 33.3 mmol,
equiv) was added. After stirring at RT for 10 min compound 9d (8.0 g,
3.3 mmol, 1 equiv) was diluted with CH Cl and added. After stirring at
2
2
3
1
3
3.3 mmol, 1 equiv) were dissolved in CH
2
2
2
4
residue (14.5 g) was purified by flash column chromatography (hexanes/
acetone 2:1) to afford compound 17 (5.25 g, 15 mmol, 75%) as yellow oil.
2
2
RT for 10 min the reaction mixture was shaken with an aqueous solution of
Na and the phases were separated. The aqueous layer was extracted
three times with CH Cl . The combined organic layers were extracted with
brine, dried with MgSO and the solvent was evaporated. The resulting
orange residue (20 g) was dissolved in MeCN and compound 5 (10.0 g,
0 mmol, 1.2 equiv) and K CO (11.5 g, 83.2 mmol, 2.5 equiv) were added.
This mixture was heated to 808C with vigorous stirring for 3 h. After
filtration the solvent was evaporated and CH Cl and aqueous NaHCO
were added. After separation of the layers the aqueous phase was extracted
twice with CH Cl . The combined organic phases were extracted with
brine, dried with MgSO and the solvent was evaporated. The residue was
1
R
f
(hexane/acetone 2:1) ˆ 0.45; H (300 MHz, CDCl
3
, 258C, TMS): d ˆ
), 1.77 ± 1.84 (m, 4H;
), 3.17 (t, J(H,H) ˆ 7.1 Hz, 2H; CH I), 4.37 ± 4.44
‡NCH ), 7.11 (s, 1H; arom. H), 7.15 (s, 1H; arom. H);
NMR (75 MHz, CDCl 258C, CDCl I), 14.03
): d ˆ 6.61 (CH
OCH CH ), 25.10 (NCH CH CH ), 29.58 (ICH CH CH ), 30.68
NCH CH ), 32.84 (ICH CH ), 47.96 (NCH ), 60.94 (COOCH ), 124.75
C5 of imidazole), 128.93 (C4 of imidazole, 135.51 (C2 of imidazole), 158.48
CO); IR (CHCl
2 2 3
S O
1.34 ± 1.46 (m, 7H; 2CH
2
3
‡OCH
2 3
CH
2
2
ICH
2
CH
2
‡NCH
2
CH
2
2
4
(
m, 4H; 2COOCH
C
2
2
1
3
3
,
3
2
4
2
3
(
(
(
(
(
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
): nÄ ˆ 2986 (s), 2939 (s), 2861 (m), 1710 (s), 1511 (w), 1473
2
2
s), 1422 (s), 1386 (s), 1304 (m), 1257 (s), 1141 (s), 1108 (s), 1071 (m),
4
À1
1
011 cm (w); HR-MS (ESI): calcd for C12
H
29
N
2
O
2
I: 350.0491; found:
subjected to flash column chromatography (hexane/acetone 2:1‡1%
‡
3
51.0568 [M‡H] .
Et
6
3
N), affording compound 14 as a yellow liquid (10.55 g, 22 mmol,
1
6%). R
f
(hexane/acetone 3:2) ˆ 0.50; H (300 MHz, CDCl
3
, 258C, TMS):
SiMe ), 1.33 (m,
), 1.77 (m, 4H; 2CH ),
Compound 18: Compound 17 (3.47 g, 9.9 mmol, 1.1 equiv) was dissolved in
3
d ˆ 0.05 (s, 9H; SiMe
3
), 0.98 (t, J(H,H) ˆ 8.2 Hz, 2H; CH
2
3
[13]
MeCN (50 mL). Then 3-nitropyrrole (1.01 g, 9.0 mmol, 1.0 equiv) and
3
4
4
H; 2CH
.29 ± 4.41 (m, 8H; 2COOCH
2
), 1.40 (t, J(H,H) ˆ 7.1 Hz, 3H; OCH
2
CH
3
2
K
2
CO
was heated to 808C for 12 h. After filtration the solvent was evaporated.
CH Cl and an aqueous solution of NaHCO were added. After separation
of the layers the aqueous layer was extracted twice with CH Cl . The
combined organic phases were extracted with brine, dried with MgSO and
the solvent was evaporated. The crude product was purified by flash
N), affording
3
(2.5 g, 18.0 mmol, 2.0 equiv) were added and the reaction mixture
2
‡2NCH
2
), 7.04 (s, 1H; arom. H), 7.12 (s, 1H;
3
3
arom. H), 7.37 (d, J(H,H) ˆ 2.2 Hz, 1H; arom. H), 7.59 (d, J(H,H) ˆ
2
2
3
1
3
2
.2 Hz, 1H; arom. H); C NMR (75 MHz, CDCl
À1.42 (SiMe ), 14.31 (OCH CH ), 17.39 (CH SiMe
CH ), 30.93 (NCH CH ), 30.98 (NCH CH ), 48.26 (NCH
1.35 (COOCH ), 63.26 (COOCH ), 112.74, 122.40, 125.00, 126.45, 129.34,
35.13, 157.13, 158.89, 159.91; IR (CHCl
3
, 258C, CDCl
), 25.97 (2 middle
), 50.22 (NCH ),
3
): d ˆ
2
2
3
2
3
2
3
4
2
2
2
2
2
2
2
6
1
2
1
2
2
column chromatography (hexane/acetone 2:1‡0.1% Et
3
3
): nÄ ˆ 3143 (m), 2985 (s), 2958 (s),
compound 18 (2.03 g, 6.07 mmol, 67%) as a yellowish oil. R
(ethyl
f
863 (s), 1711 (s), 1540 (s), 1511 (s), 1473 (s), 1422 (s), 1385 (s), 1371 (s),
1
acetate) ˆ 0.40; H (300 MHz, CDCl
3
, 258C, TMS): d ˆ 1.33 ± 1.46 (m, 7H;
À1
318 (s), 1252 (s), 1151 (s), 1107 (s), 1082 (s), 925 cm (m); HR-MS (ESI):
3
2
CH
2
‡OCH
2
CH
3
), 1.79 ± 1.82 (m, 4H; 2NCH
2
CH
2
), 3.88 (t, J(H,H) ˆ
calcd for C22
H
34
N
4
O
6
Si: 478.2248; found: 479.2324 [M‡H]‡.
7
.1 Hz, 2H; N(Py)CH
2
), 4.40 (m, 4H; 2COOCH
2
‡N(Im)CH
2
), 6.54 (t,
3
3
Compound 15: Obtained analogously to compound 11d, using compound
1
3
J(H,H) ˆ 2.7 Hz, 1H; H4 of Py), 6.70 (dd, J(H,H) ˆ 1.8, 2.7 Hz, 1H; H5 of
3
3
0d (8.6 g, 15.7 mmol, 1 equiv) and TBAF (47.1 mL, 1m in THF, 47.1 mmol,
equiv), affording the tetrabutylammonium salt of the deprotected acid
Py), 7.06 (d, J(H,H) ˆ 0.9 Hz, 1H; H5 of Im), 7.15 (d, J(H,H) ˆ 1.2 Hz,
3 13
1H; H4 of Im), 7.50 (t, J(H,H) ˆ 2.1 Hz, 1H; H2 of Py); C NMR
(75 MHz, CDCl 258C, CDCl CH ), 25.95
): d ˆ 14.32 (OCH
(NCH CH CH ), 30.64 (NCH CH ), 30.95 (NCH CH ), 48.20 (N(Im)CH ),
50.63 (N(Py)CH ), 61.38 (COOCH ), 105.52 (C5 of Py), 121.14 (C2 or C4 of
(
9.25 g, 13.4 mmol, 85%). A part of it (2.00 g, 2.9 mmol, 1 equiv) was
coupled with DCC (2.75 mL, 1m in CH Cl , 2.75 mmol, 0.95 equiv), HOBt
588 mg, 4.35 mmol, 1.5 equiv), NH (7.0 mL, 0.5m in dioxane, 3.48 mmol,
.2 equiv) and DIEA (1 mL, 5.80 mmol, 2 equiv), affording compound 15
1.2 g, 2.68 mmol, 92%) as colorless foam. R
(hexane/acetone 2:3) ˆ 0.38;
3
,
3
2
3
2
2
2
2
2
2
2
2
2
2
(
1
3
2
2
Py), 121.35 (C4 or C2 of Py), 125.00 (C5 of Im), 129.38 (C4 of Im), 135.87
(C3 of Py or C2 of Im), 136.58 (C2 of Im or C3 of Py), 158.90 (CO); IR
(
f
1
H
NMR (300 MHz, DMSO, 258C, DMSO): d ˆ 1.16 (m, 4H;
(CDCl
3
): nÄ ˆ 2943 (m), 2863 (w), 1710 (s), 1532 (m), 1514 (m), 1489 (s), 1473
2
NCH
2
CH
2
CH
2
), 1.25 (t, 3J(H,H) ˆ 7.1 Hz, 3H; OCH
2 3
CH ), 1.40 (s, 9H;
(m), 1422 (s), 1385 (m), 1365 (m), 1294 (s), 1257 (m), 1155 (m), 1129 (m),
Chem. Eur. J. 2003, 9, 3353 ± 3366
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3365

P. B. Dervan and A. Heckel
FULL PAPER
À1
1
3
072 cm (m); HR-MS (ESI): calcd for C16
H
22
N
4
O
4
: 334.1641; found:
Janssen, T. Durussel, U. K. Laemmli, Mol. Cell 2000, 6, 999 ± 1011;
d) K. Maeshima, S. Janssen, U. K. Laemmli, EMBO J. 2001, 20, 3218 ±
‡
35.1716 [M‡H] .
3
228; e) M. P. Gygi, M. D. Ferguson, H. C. Mefford, P. L. Kevin, C.
Compound 19: Compound 18 (0.9 g, 2.7 mmol, 1 equiv) was dissolved in
O×Day, P. Zhou, C. Friedman, G. van den Engh, M. L. Stobowitz, B. J.
Trask, Nucleic Acids Res. 2002, 30, 2790 ± 2799.
EtOAc and Boc
purging the solution with Ar, Pd/C (311 mg, 10%) was added. This mixture
was stirred under a H atmosphere (35 bar) at RT for 3.5 h. Then it was
filtered and the solvent was evaporated. Purification by flash column
N) afforded compound 19
(hexane/acetone 1:1) ˆ 0.56;
), 1.43 (t,
), 1.49 (s, 9H; tBu), 1.70 ± 1.80 (m, 4H;
2
O (617 mg, 2.83 mmol, 1.05 equiv) was added. After
[
2] V. C. Rucker, S. Foister, C. M. Melander, P. B. Dervan, J. Am. Chem.
Soc. 2003, 125, 1195 ± 1202.
2
[
3] An obvious limitation of this model is that pairing of a dip (as in A ¥ T)
with another dip (as in Py/Im, this is the second situation from the top
in the mismatch column in Figure 1) is mechanically possible in this
model. However, for the sake of simplicity, we did not want to resort
to more difficult recognition surfaces but chose to phrase the rules
accordingly. However, after a slight modification of the rules, the
model can easily be extended to also illustrate the so-called 1:1
binding mode (cf. A. R. Urbach, P. B. Dervan, Proc. Natl. Acad. Sci.
USA 2001, 98, 4343 ± 4348).
chromatography (hexane/acetone 2:1‡0.1% Et
3
as a colorless oil (0.96 g, 2.37 mmol, 88%). R
f
1
H (300 MHz, CDCl
3
, 258C, TMS): d ˆ 1.30 (m, 4H; 2CH
2
3
J(H,H) ˆ 7.1 Hz, 3H; OCH
2 3
CH
3
2
2
(
NCH
2
CH
2
), 3.76 (t, J(H,H) ˆ 7.1 Hz, 2H; N(Py)CH
2
), 4.34 ± 4.44 (m, 4H;
3
COOCH
2
‡N(Im)CH
2
), 5.85 (t, J(H,H) ˆ 2.2 Hz, 1H; C4 of Py), 6.24
3
brs, 1H; NH), 6.43 (t, J(H,H) ˆ 2.7 Hz, 1H; C5 of Py), 6.87 (brs, 1H; C2
13
of Py), 7.04 (s, 1H; C5 of Im), 7.14 (s, 1H; C4 of Im); C NMR (75 MHz,
CDCl , 258C, CDCl CH ), 26.14 (NCH CH CH ), 26.26
CH CH ), 28.46 (C(CH ), 31.10 (2 NCH CH ), 48.38 (N(Im)CH ),
9.78 (N(Py)CH ), 61.36 (COOCH ), 79.56 (C(CH ), 99.86 (C4 of Py),
09.91 (C2 or C5 of Py), 118.52 (C5 or C2 of Py), 122.58 (C3 of Py), 125.08
3
3
): d ˆ 14.37 (OCH
2
3
2
2
2
[
[
4] In a mixture of two different unconnected polyamide strands (with for
example four residues each) with DNA two 1:1 complexes and three
(
NCH
2
2
2
3
)
3
2
2
2
4
1
(
(
1
(
2
2
3 3
)
2
:1 complexes can potentially be formed. In 2:1 complexes the
polyamides either bind as a homodimer (if two molecules of the same
strand bind side by side) or a heterodimer.
C5 of Im), 129.35 (C4 of Im), 135.93 (C2 of Im), 157.10 (CO (Boc)), 158.93
COOEt); IR (CHCl
): nÄ ˆ 2956 (m), 2863 (w), 1712 (s), 1577 (w), 1540 (m),
491 (m), 1422 (m), 1390 (m), 1369 (m), 1312 (w), 1250 (s), 1165 (s), 1120
m), 1072 (w), 1059 (w), 1019 cm
21 32 4 4
C H N O
3
5] a) M. Mrksich, M. E. Parks, P. B. Dervan, J. Am. Chem. Soc. 1994, 116,
7983 ± 7988; b) M. Mrksich, P. B. Dervan, J. Am. Chem. Soc. 1993, 115,
9892 ± 9899.
À1
(w); HR-MS (ESI): calcd for
: 404.2424; found: 405.2499 [M‡H]‡.
[
[
[
6] S. E. Swalley, E. E. Baird, P. B. Dervan, J. Am. Chem. Soc. 1999, 121,
Compound 20: Compound 19 (291 mg, 0.72 mmol, 1 equiv) was dissolved
1
113 ± 1120.
in a solution of NaOH in MeOH/H
equiv). After shaking at 378C for 12 h the reaction mixture was
lyophilized, affording compound 20 as yellowish foam (280 mg,
.70 mmol, 98%). This compound was always freshly prepared before
2
O 1:1 (3.6 mL, 0.2m, 0.72 mmol,
7] J. M. Belitsky, D. H. Nguyen, N. R. Wurtz, P. B. Dervan, Bioorg. Med.
Chem. 2002, 10, 2767 ± 2774.
8] See for example: a) G. Burckhardt, H. Simon, E. Birch-Hirschfeld, L.
Kittler, S. K. Sharma, J. W. Lown, C. Zimmer, J. Biomol. Struct. Dyn.
1
a
0
1
the solid-phase synthesis. H NMR (300 MHz, DMSO, 258C, DMSO): d ˆ
2
002, 19, 1101 ± 1109; b) C. C. O×Hare, D. Mack, M. Tandon, S. K.
1
(
5
1
.18 (m, 4H; 2CH
2
), 1.41 (s, 9H; tBu), 1.59 ± 1.64 (m, 4H; 2NCH
2
CH
2
), 3.70
),
Sharma, J. W. Lown, M. L. Kopka, R. E. Dickerson, J. A. Hartley,
Proc. Natl. Acad. Sci. USA 2002, 99, 72 ± 77; c) W. A. Greenberg, E. E.
Baird, P. B. Dervan, Chem. Eur. J. 1998, 4, 796 ± 805; d) Y. H. Chen,
Y. W. Yang, J. W. Lown, J. Biomol. Struct. Dyn. 1996, 14, 341 ± 355;
e) Y. H. Chen, J. W. Lown, Biophys. J. 1995, 68, 2041 ± 2048; f) Y. H.
Chen, J. W. Lown, J. Am. Chem. Soc. 1994, 116, 6995 ± 7005; g) M.
Mrksich, P. B. Dervan, J. Am. Chem. Soc. 1994, 116, 3663 ± 3664; h) M.
Mrksich, P. B. Dervan, J. Am. Chem. Soc. 1993, 115, 9892 ± 9899.
9] B. Olenyuk, C. Jitianu, P. B. Dervan, J. Am. Chem. Soc. 2003, 125,
3
3
t, J(H,H) ˆ 7.1 Hz; 2H, NCH
2
), 4.42 (t, J(H,H) ˆ 7.1 Hz; 2H; NCH
2
.79 (s, 1H; arom. H), 6.46 (s, 1H; arom. H), 6.70 (s, 1H; arom. H), 6.74 (s,
1
3
H; arom. H), 7.06 (s, 1H; arom. H); C NMR (75 MHz, DMSO, 258C,
CH CH ), 25.79 (NCH CH CH ), 28.37
), 31.16 (NCH CH ), 46.76 (NCH ), 48.72
), 99.86, 108.77, 118.01, 121.78, 124.48, 125.47,
44.93, 152.99, 162.09; HR-MS (ESI): calcd for C19 : 376.2111;
DMSO): d ˆ 25.68 (NCH
2
2
2
2
2
2
(
C(CH
3
)
3
), 30.93 (NCH
2
CH
2
2
2
2
(
1
NCH ), 77.22 (C(CH )
2
3 3
28 4 4
H N O
‡
found: 399.2011 [M‡Na] .
Construction of plasmid DNA: To obtain the plasmid pAH2, the
oligonucleotide strands printed in bold face in Figure 6 were hybridized
and then ligated (Roche rapid ligation kit) into linearized pUC19[17]
BamHI/HindIII) using T4 DNA ligase. The resultant construct was used
to transform JM109 cells (Promega). Ampicillin-resistant white colonies
were selected from Luria-Bertani medium-agar plates containing ampicil-
lin (50 mg mL ), IPTG (120 mg mL ) and XGAL (40 mg mL ) and
grown in Luria-Bertani medium. Then the plasmid was isolated using the
Qiagen HiSpeed Plasmid Purification Kit. The sequence was verified by
[
4
741 ± 4745.
[
[
10] C. Melander, V. Rucker, P. B. Dervan, unpublished results.
11] a) For solid-phase synthesis using the Boc strategy see: E. E. Baird,
P. B. Dervan, J. Am. Chem. Soc. 1996, 118, 6141 ± 6146; b) for Fmoc
strategy see: N. R. Wurtz, J. M. Turner, E. E. Baird, P. B. Dervan, Org.
Lett. 2001, 3, 1201 ± 1203; c) for a solid-phase synthesis strategy of
polyamides with a variety of different tail residues see ref. [7].
12] T. Nishiguchi, K. Kawamine, T. Ohtsuka, J. Org. Chem. 1992, 57, 312 ±
(
À1
À1
À1
[
[
[
3
16.
dideoxy
sequencing
using
the
primers
PBD1f
(5'-
13] B. L. Bray, P. H. Mathies, R. Naef, D. R. Solas, T. T. Tidwell, D. R.
Artis, J. M. Muchowski, J. Org. Chem. 1990, 55, 6317 ± 6328.
14] Iodide 17 is a remarkably stable compound which can for example be
heated under the conditions of the alkylation (also in the presence of
CTGCGCAACTGTTGGGAAGGG-3') and PBD1r (5'-GGGCAGT-
GAGCGCAACGCAAT-3'). The plasmid pDEH9 had been prepared in
a similar way by S. E. Swalley and D. E. Herman.
Preparation of 3'-end-labeled restriction fragments: Following a literature
OPPh ) without the nucleophile for several days without any signs of
decomposition.
3
[
20]
procedure, the plasmids pDEH9 and pAH2 were linearized with EcoRI
and PvuII and then treated with Klenow enzyme, deoxyadenosine 5'-[a-
[15] For the synthesis of compounds 21 and 22 see ref. [11a]. Compound 24
was obtained according to ref. [2]. Compound 25 can be obtained
according to ref. [16].
[16] W. S. Wade, M. Mrksich, P. B. Dervan, J. Am. Chem. Soc. 1992, 114,
8783 ± 8794.
3
2
32
P]triphosphate and thymidine 5'-[a- P]triphosphate. After separation on
a nondenaturing polyacrylamide gel the desired band was visualized by
autoradiography and isolated. Chemical sequencing reactions were per-
formed according to published methods.[
21]
[
[
17] C. Yanisch-Perron, J. Vieira, J. Messing, Gene 1985, 33, 103 ± 119.
18] For examples see ref. [8b] and S. White, E. E. Baird, P. B. Dervan,
Chem. Biol. 1997, 4, 569 ± 578; M. E. Parks, E. E. Baird, P. B. Dervan,
J. Am. Chem. Soc. 1996, 118, 6153 ± 6159.
Acknowledgement
We are grateful to the National Institutes of Health for research support, to
the Alexander von Humboldt-Foundation (Germany) for a Feodor Lynen
Fellowship for A.H. and to Dr. Bogdan Olenyukfor helpful discussions.
[19] H. E. Gottlieb, V. Kotlyar, A. Nudelman, J. Org. Chem. 1997, 62,
7512 ± 7515.
[20] J. W. Trauger, P. B. Dervan, Methods Enzymol. 2001, 340, 450 ± 466.
[
21] a) B. L. Iverson, P. B. Dervan, Nucleic Acids Res. 1987, 15, 7823 ± 7830;
b) A. M. Maxam, W. S. Gilbert, Methods Enzymol. 1980, 65, 499 ± 560.
[
1] a) P. B. Dervan, Bioorg. Med. Chem. 2001, 9, 2215 ± 2235; b) P. B.
Dervan, R. W. B¸rli, Curr. Opin. Chem. Biol. 1999, 3, 688 ± 693; c) S.
Received: January 29, 2003 [F4784]
3366
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3353 ± 3366