Solid-phase synthesis of asymmetrically branched sequence-defined poly/oligo(amidoamines)

Article abstract of DOI:10.1021/jo202561k

We present for the first time the synthesis of asymmetrically branched sequence-defined poly/oligo(amidoamines) (PAAs) using solid-phase synthesis with the capability of introducing diversity at the side chains. We introduce two new versatile (diethylenet

Full text of DOI:10.1021/jo202561k

Article
pubs.acs.org/joc
Solid-Phase Synthesis of Asymmetrically Branched Sequence-
Defined Poly/Oligo(amidoamines)
Felix Wojcik, Simone Mosca, and Laura Hartmann*
Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am Muhlenberg 1, 14476 Potsdam, Germany
̈
S
* Supporting Information
ABSTRACT: We present for the first time the synthesis of asymmetrically branched sequence-defined poly/oligo-
(amidoamines) (PAAs) using solid-phase synthesis with the capability of introducing diversity at the side chains. We introduce
two new versatile (diethylenetriamine) building blocks for solid-phase synthesis bearing Fmoc/Boc and Fmoc/Alloc protecting
groups expanding recently used Fmoc/Boc protecting group strategy for linear PAAs to an Fmoc/Alloc/Boc strategy. This allows
for orthogonal on-resin cleavage of Fmoc and Alloc protecting groups during solid-phase synthesis of PAAs with backbones
differing in chain length and sequence. With these structures we then demonstrate the potential for generating asymmetrical
branching by automated multiple on-resin cleavage of Alloc protecting groups as well as the introduction of side chains varying in
length and number. Such systems have high potential as nonviral vectors for gene delivery and will allow for more detailed
studies on the correlation between the degree of branching of PAAs and their resulting biological properties.
could also increase cytotoxicity, the degree of branching, as well
as the number of cationic functionalities along the branches,
seems to be of great importance for the design of new and
improved PAA carrier systems.18
INTRODUCTION
■
Recently, a novel class of peptidomimetic oligocations based on
poly(amidoamines) have been recognized for their significant
potential as nonviral vectors in gene delivery.1−4 In contrast to
classic polycationic carrier systems,5−8 these are highly defined
synthetic systems that allow for a step-by-step correlation
between their chemical structure and the resulting biological
properties of the carrier.9,10 We previously presented a novel
approach for the synthesis of sequence-defined linear poly/
oligo(amidoamines) (PAAs) with high efficiency using solid-
phase synthesis and showed their use as nonviral vectors in
gene delivery.11−13 In order to overcome side reactions
observed during solid support synthesis, Schaffert et al.14
improved on our approach15,16 by exploiting Fmoc as a
temporary protective group for Boc-protected triethylenetetr-
amine acids and tetraethylenepentamine acids. They also used
this process to illustrate the synthesis of symmetrical branched
structures by using Fmoc-Lys(Fmoc)-OH. Furthermore, they
were able to show that the introduction of branching points
leads to improved carrier properties, resulting in more efficient
transfection reagents as is also known for other branched
polycationic systems17 like poly(ethyleneimine)18 and poly-
disperse PAAs19 or dendritic PAAs.20 This effect is often
explained by cationic functionalities participating in the so-
called proton sponge effect enabling an effective release of the
cargo.21,22 Since at the same time these cationic functionalities
So far, only symmetrically branched PAAs have been
obtained by applying Fmoc-Lys(Fmoc)-OH as a building
block.1,14 After Fmoc cleavage, the main chain can now grow in
two directions leading to a symmetrical branching point. Here
we now present a new set of building blocks for PAA synthesis
allowing for the first time separate building of the main and side
chains on the solid phase. We refer to these systems as
asymmetrically branched sequence-defined PAAs. Suitable
building blocks need to introduce a protective group that can
be cleaved during solid-phase synthesis and which is orthogonal
to Fmoc.23 This protecting group strategy24 will give access to a
controlled number and length of side chains as well as main and
side chains of different chemical compositions.
We chose to expand on the already known Fmoc/Boc
protecting group strategy14 to an Fmoc/Alloc/Boc strategy for
the straightforward synthesis of asymmetric PAAs. The Fmoc
protecting group is used as a temporary protecting group
during synthesis of the main chain, while the Boc protecting
group is only cleaved after complete synthesis of the desired
Received: December 13, 2011
Published: April 9, 2012
© 2012 American Chemical Society
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Figure 1. Novel approach for the synthesis of asymmetrically branched PAAs referring to a Fmoc/Alloc/Boc strategy.
Scheme 1. Building Block Synthesis Starting from Diethylenetriamine Leading to ADS 7 and BDS 8
PAA molecule. The Alloc protecting group23,25 now allows for
selective cleavage on resin and the introduction of side chains
or branching points leading to asymmetrically branched
sequence-defined PAAs (Figure 1).
general, building blocks should also consist of a backbone unit
containing a protected amine and free carboxylate functionality
with the capability of introducing additional functionalities into
the backbone. This work specifically focuses on introducing a
secondary amine carrying either a Boc or Alloc protecting
group.
In general, we are interested in a synthetic approach for
building blocks not only suitable for the Fmoc/Boc/Alloc
approach but also for straightforward synthesis of various
building blocks. Therefore, we chose an approach starting from
a simple triamine, diethylenetriamine, and differentiating the
RESULTS AND DISCUSSION
■
Building blocks suitable for PAA solid-phase synthesis have to
fulfill certain requirements: they have to be accessible in large
quantities and high purity and have to be compatible with
standard solid-phase peptide synthesis (SPPS) coupling
protocols; e.g., they need to be soluble in DMF or NMP. In
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Figure 2. RP-HPLC analysis (5−50% MeCN in 60 min) of compound 14 referring to different allyl scavengers during Alloc deprotection. Sequential
Alloc deprotection and BDS coupling using phenylsilane (A) and N,N′-dimethlybarbituric acid (B) as allyl scavenger.
three amine groups by a multistep protecting protocol, which
can then be coupled to a diacid and produce the corresponding
dimer building block referring to our original diacid and
diamine building block approach.11 During this synthesis,
different protecting groups as well as functional side groups can
be introduced at the secondary amine to give straightforward
access to a library of building blocks from a key intermediate
presented here for the first time.
For this key intermediate, the two primary amine groups of
diethylenetriamine were differentiated by quantitative protec-
tion of just one amine group exploiting the bulky
triphenylmethyl group in the presence of an excess of
diethylenetriamine26 to give the corresponding monotrityl-
protected triamine 1. Then ethyl trifluoroacetate was used to
protect the remaining primary amine27 of compound 1 to give
the asymmetrical protected precursor 2 on a scale of up to 120
g (Scheme 1).
Compound 2 as a novel key intermediate can now be
functionalized at the secondary amine with various protecting
groups as well as through coupling of functional side chains for
conjugaton chemistry28 or the attachment of biological ligands
or fluorescent labels. Here, for the synthesis of the Fmoc/Boc
and Fmoc/Alloc building blocks, it was selectively functional-
ized with either a Boc or an Alloc group by reaction with Alloc-
Cl or Boc anhydride providing compound 3 and 4. The Boc-
and Alloc-protected triamines were then converted into the
final building blocks: First, the TFA group was converted to an
Fmoc moiety using a one-pot protocol giving compound 5 and
6. Then the trityl group was selectively cleaved, and the
corresponding primary amine group was coupled to succinic
anhydride affording the final building blocks for the Fmoc solid-
phase synthesis, ADS 7 and BDS 8 with an overall yield of 50%
and 43%, respectively. The complete synthesis can be carried
out in large scale resulting in up to 41 g of final building block
in one synthesis. Furthermore, no chromatographic purification
is needed. At this point, it is important to describe our
nomenclature for these groups. Our nomenclature for novel
PAA building blocks indicates the side group, diamine, and
diacid components: The two building blocks are functionalized
with Alloc or Boc on the secondary amine and contain
Diethylentriamine as the diamine unit and Succinic acid as the
diacid unit.
During trityl cleavage, we observed a shielding of the trityl
protecting group, probably by the other protecting groups in
the molecule. This effect is also known for N-trityl α-amino
acid esters and requires a higher concentration of TFA for
quantitative trityl cleavage.29 Here, higher concentrations of
TFA (>3%) in DCM had little impact on the stability of the
Alloc-containing compound 5, but already 4% TFA in DCM
showed about 15% cleavage of the Boc protecting group of
compound 6 after only 30 min. Therefore, a new protocol for
the cleavage of shielded trityl protecting groups in the presence
of Boc was necessary. We found that a mixture of acetic acid
and trifluoroethanol 1:1 at 60 °C for 1 h resulted in exclusive
deprotection of the trityl group without formation of the Boc-
deprotected byproduct (see the Supporting Information). To
our knowledge, this is a new method for the deprotection of
such shielded N-trityl groups in the presence of Boc protecting
groups.
After the complete building block synthesis, we proved by
NMR and HPLC that ADS and BDS were obtained in very
high purity (>99%) without referring to any chromatographic
purification method. The building blocks were then directly
applied for fully automated solid-phase PAA synthesis using an
automated standard peptide synthesizer. In a first set of
experiments, five backbones of different length and varying
sequences containing the Boc as well as Alloc building block
were synthesized, showing the suitability of the new building
blocks for PAA solid-phase synthesis. All backbones were
synthesized following Fmoc amino acid coupling protocols
using double coupling with 5 equiv of building blocks 7 or 8
and coupling times of 1 h with HATU as an activation agent30
(for the backbone coupling pattern, see Figure 4, Supporting
Information). Although ADS and BDS are significantly longer
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Table 1. Overview of PAA Backbones and Asymmetrically Branched PAAs Including MALDI TOF MS Analysis and Purity by
RP-HPLC Analysis (for HPLC and MS Spectra See the Supporting Information)
*1
The most abundant ion adduct is shown here; a detailed MALDI analysis and spectra can be found in the Experimental Section and in the
*2
Supporting Information. According to previously determined purity of the Fmoc-containing compound, followed by complete Fmoc-deprotection
shown with H NMR.
1
than α- or β-amino acids or even pseudoprolines31 used in
SPPS, solid-phase coupling gave PAA backbones in very high
purity of up to 15 dimer building blocks (Figure 2a, Table 1).
The backbones 9−13 were synthesized on solid support in
purities of >95% for the homo oligomer consisting of 5 ADS
building blocks (PAA 9) and purities of 83% for a 15mer
bearing 10 BDS building blocks and 5 ADS building blocks
(PAA 13), respectively. The decrease in purity for PAA chains
bearing more then 10 repeating units can be attributed to a
decrease in coupling efficiency for longer PAA chains; similar
trends are known from standard SPPS. Boc as well as Alloc
protecting groups are stable during backbone synthesis; thus,
no undesired branching during backbone synthesis was
observed, and different chain lengths as well as different
sequences of Alloc and Boc building blocks within the chain
were obtained (Table 1).
After synthesis of the desired PAA backbone, capping of the
N-terminal side for the following side-chain elongation proved
to be essential in order to avoid further backbone growth. For
this, we exploited acetic anhydride. Notably, different acid
bearing compounds can also be applied for the capping, e.g.,
allowing for the introduction of lipids, different temporary
protecting groups, hydrophobic dyes, or the attachment of
polymer chains.
This first set of backbones was then applied to test for the
efficiency of Alloc cleavage and the elongation of side chains at
the introduced branching points. The Alloc protecting groups
introduced into the main chain were cleaved orthogonally by
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Figure 3. RP-HPLC analysis (5% to 50% MeCN in 60 min) of PAA backbone 10, asymmetrically branched PAA 16 bearing 12 bearing block and
asymmetrically branched PAA 24 bearing 16 building blocks with 2× side-chain elongation.
the use of palladium(0) and an appropriate scavenger for the
generated allyl species (Figure 2), while the PAA chain was still
attached to the solid support. In order to achieve a fully
automated synthesis, this reaction was performed on a peptide
synthesizer.32 In a first attempt to cleave Alloc groups on solid
support, we observed incomplete deprotection and the
formation of undesired side products. We also showed that
for the PAA systems, the secondary amines generated after
Alloc cleavage could also serve as a nucleophile during Alloc
cleavage resulting in undesired byproduct. This was especially
critical for PAA backbones containing more than 2 Alloc
building blocks (see PAA backbone 9, Figure 2) when used in
the presence of phenylsilane, one of the most common allyl-
scavenger in SPPS (see Figure 2a). In addition, tandem
deprotection/coupling33 using DABCO, EDC, HOBT, and
BDS in DCM proved to be inefficient and resulted in allylated
byproduct. In order to avoid the formation of allylated
byproduct, we used N,N′-dimethylbarbituric acid as the
scavenger34 (see Figure 2b). With these conditions, full
deprotection of all Alloc groups was achieved releasing free
secondary amines within the backbone while retaining
backbone integrity.
Side-chain elongation was performed by applying the same
fully automated coupling conditions used for the backbone
synthesis. All five PAA systems were used for the introduction
of side chains (Table 1) at the secondary amine groups released
after Alloc cleavage. Therefore, BDS 8 was coupled to the PAA
backbones resulting in branched PAAs with different numbers
of side chains depending on the number of Alloc building
blocks introduced during main-chain synthesis. Control over
the chemical structure was obtained by ensuring close to
complete conversion in every reaction step as was proven by
NMR, HPLC, and MS (table 1, Figure 3b, Supporting
Information).
15 (14 after Fmoc deprotection, for HPLC spectra see the
Supporting Information) showing similar purity as PAA 14.
At this point, we were able to install three (PAA 18 and 19),
four (PAA 17, 18, 20, 21), and five (PAA 14, 15, 22, 23) side
chains simultaneously with different spacing along the PAA
backbone: no spacing (PAA 14 and 15), one BDS building
block (PAA 16 and 17), and two BDS building blocks (PAA
18-23). All of these systems present a first generation branched
PAA structure bearing one 1,2-diaminoethane motif35 in the
side chain.
In order to now allow for a further growth of side chains or
attachment of additional moieties, Fmoc end groups in the side
chain have to be cleaved. Again, this step was performed fully
automated on the peptide synthesizer. Analysis by NMR and
MS proved successful cleavage of all Fmoc protecting groups,
now allowing for further coupling reactions to the primary
amine groups in the side chains (Table 1).
In order to show that further elongation was feasible, we
chose the PAA backbone 10 containing 4 ADS building blocks
and 4 BDS building blocks (see Figure 3). After the successful
synthesis of the first-generation branched PAA 16 (see Figure
3) and Fmoc deprotection of the side chains (PAA 17), a
second Boc building block BDS was coupled to the PAA side
chains (see Figure 3, PAA 24) followed by Fmoc deprotection
to give the unprotected PAA 25. Analysis via RP-HPLC, NMR,
and MS proved the successful synthesis of a second generation
branched PAA system now presenting two repeating units of
the BDS motif in the side chains. As was observed for longer
linear PAA chains, we also see a decrease in product purity to
about 80% directly after resin cleavage, indicating a loss of
coupling efficiency for larger PAA structures. During LC/MS
analysis, a deletion sequence of one Fmoc-BDS-moiety after the
last coupling step could be identified as the major impurity
(about 9%) in PAA 24. Here, this could be attributed to the
polarity of the side chains rather than the chain length as was
indicated by high purities of PAAs bearing all α-amino acid side
chains of 90% (unpublished data).
The purity of all asymmetrically branched PAAs was
determined by integration of the HPLC UV spectra at 214
nm. After introducing the asymmetrical branches, we proved
that all peaks in the HPLC UV spectra contain Fmoc moieties
by connecting a fluorescence detector (Ex 259, Em 311) to the
HPLC system. Furthermore, we used LC/MS analysis of the
Fmoc-protected stages, which show exclusively the MS of the
desired asymmetrically branched PAAs within the main peak
(for LC/MS spectra, see the Supporting Information). Finally,
we were able to compare these purities obtained by RP-HPLC
with those obtained by strong cation exchange HPLC of PAA
All PAAs shown here are finally purified after cleavage from
the resin and precipitation from diethylether. This is one
prerequisite of our synthetic strategy as we want to have access
to highly pure samples by an optimized synthetic procedure
rather then by elaborate purification of a mixture of products,
e.g., by preparative HPLC. Nevertheless, if highly pure samples
(purity >95%) are required for special future biophysical and in
vitro characterization, further purification of all presented
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Trityl chloride (69.7 g, 250 mmol) was dissolved in 250 mL of
DCM and added dropwise to diethylenetriamine solution at 0
°C. After 2 h, the mixture was slowly warmed to room
temperature and stirred for an additional 3 h. After 5 h reaction
time, the mixture was concentrated under reduced pressure to 1
L and washed with 3 × 500 mL aqueous satd NaHCO3
solution. The organic layer was dried over MgSO4, filtered,
and evaporated under reduced pressure to give 1 as a white,
hygroscopic solid (86 g, 250 mmol) in quantitative yield: mp =
asymmetrically branched PAA systems is possible via
preparative RP-HPLC of the Fmoc protected structures.
In general, we showed in this work that solid-phase synthesis
provides an unique tool for the synthesis of highly defined
asymmetrically branched poly/oligo(amidoamines) within the
molecular weight range of small polymers (20 repeating
units).36,37 Further optimization of this method could lead to
larger structures, but due to the solid-phase approach there is
an upper limit as already known, e.g., from peptide chemistry.38
This could be overcome for structures >10 kDa by employing
chemoselective ligation28 or conjugation chemistry resulting in
monodisperse systems. Furthermore, the poly/oligo-
(amidoamines) can be transformed into macromonomers,39
thus providing access to larger polymers with highly controlled
repeating patterns.
1
105−110 °C; IR (film) ν 3282, 1723, 1678, 1147 cm−1; H
NMR (400 MHz, CDCl3) δ 7.48 (dd, J1=J2= 7.7 Hz, 6H), 7.27
(t, J = 7.7 Hz, 6H), 7.17 (t, J = 7.2 Hz, 3H), 2.75−2.72 (m,
4H), 2.60−2.57 (m, 2H), 2.29−2.26 (m, 2H); 13C NMR (100
MHz, CDCl3) δ 146.1, 128.6, 127.7, 126.2, 70.7, 52.1, 49.9,
43.2, 41.6; ESI-HRMS calcd for C23H28N3 [M + H]+ 346.2283,
found 346.2272.
Synthesis of 2,2,2-Trifluoro-N-(2-((2-(tritylamino)ethyl)amino)-
ethyl)acetamide (2). Compound 1 (86 g, 250 mmol) was dissolved
in 500 mL of THF and cooled to 0 °C. Under inert atmosphere, ethyl
trifluoroaceate (31.3 mL, 263 mmol) was added slowly to the reaction
mixture, and then the solution was stirred overnight. The solvent was
evaporated under reduced pressure, and the resulting crude product
was recrystallized from toluene. Compound 2 (99 g, 224 mmol) was
isolated as colorless crystals with a yield of 90%: mp = 118−120 °C; IR
(film) ν 3349, 2849, 1706, 1147 cm−1; 1H NMR (400 MHz, CDCl3) δ
7.49−7.47 (m, 6H), 7.31−7.24 (m, 6H), 7.21−7.16 (m, 3H), 3.40−
3.34 (m, 2H), 2.75 (t, J = 5.7 Hz, 2H), 2.70 (t, J = 6 Hz, 2H), 2.29 (t, J
= 6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 157.7, 157.3, 156.9,
156.6, 145.9, 128.5, 127.8, 126.3, 120.2, 117.3, 114.5, 111.6, 70.7, 49.4,
47.1, 43.2, 38.9; ESI-HRMS calcd for C25H27F3N3O [M + H]+
442.2106, found 442.2111.
CONCLUSION
■
We introduced a new strategy toward the synthesis of
asymmetrically branched PAAs for solid support synthesis.
Therefore, a new versatile approach for the synthesis of dimer
building blocks applicable for fully automated PAA synthesis
was developed. Following this procedure, we synthesized a set
of two novel building blocks, ADS and BDS, introducing either
Boc or for the first time Alloc-protected amine groups within
the PAA main chain. The Alloc protecting group now allows for
multiple on-resin cleavage and release of secondary amines
within the PAA backbone. The resulting secondary amine
groups were then used for introduction and elongation of side
chains by the addition of Boc building blocks (BDS). Here, also
diversification of the side chains is possible by coupling to the
secondary amines, e.g., with other building blocks such as
amino acids. All systems are sequence-defined thus allowing for
the precise control of the number and positioning of side chains
along the PAA backbone. Furthermore, the complete synthesis
including growth of the main chain, cleavage of the Alloc
protecting groups, as well as elongation of the side chains was
performed continuously and fully automated on a standard
peptide synthesizer.
Synthesis of Allyl (2-(2,2,2-Trifluoroacetamido)ethyl)(2-
(tritylamino)ethyl)carbamate (3). Compound 2 (44 g, 100 mmol)
and triethylamine (27.9 mL, 200 mmol) were dissolved in 500 mL of
DCM under inert atmosphere. The solution was cooled to 0 °C, and
allyl chloroformate (11.2 mL, 105 mmol) was added dropwise. Then
the reaction mixture was slowly warmed to room temperature and
stirred for an additional 4 h. Afterward, the mixture was evaporated
under reduced pressure, and the crude product was redissolved in 500
mL of diethyl ether. The organic layer was washed twice with 400 mL
satd sodium bicarbonate solution, dried over NaSO4, and evaporated
under reduced pressure. Compound 3 (47.8 g, 91 mmol) was isolated
as a hygroscopic white solid with a yield of 91%. The crude product
was used for the next reaction without further purification: mp = 98−
This synthetic approach now allows the synthesis of a library
of differently multiple branched PAAs varying in the number
and distance of branching points within the PAA backbone as
well as chain length or chemical composition of main and side
chain. In the future, these systems will be systematically
analyzed for their potential as nonviral vectors as well as a novel
platform for polymer bioconjugates for drug-targeting
applications.
1
100 °C; IR (film) ν 3286, 1722, 1678, 1146 cm−1; H NMR (400
MHz, CDCl3) δ 7.80−7.72 (bs, 1H), 7.37−7.32 (m, 6H), 7.22−7.15
(m, 6H), 7.13−7.07 (m, 3H), 5.80−5.70 (m, 1H), 5.20−5.08 (m, 2H),
4.48 (d, J = 5.1 Hz, 2H), 3.43−3.33 (m, 4H), 3.27 (t, J = 6.2 Hz, 2H),
2.30−2.23 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 158.1, 157.7,
157.4, 157.3, 145.7, 132.2, 128.4, 127.9, 126.4, 125.3, 118.2, 117.2,
114.3, 70.8, 66.7, 48.4, 46.4, 42.3, 40.3; ESI-HRMS calcd for
C29H31F3N3O3 [M + H]+ 526.2318, found. 526.2333.
Synthesis of tert-Butyl (2-(2,2,2-Trifluoroacetamido)ethyl)(2-
(tritylamino)ethyl)carbamate (4). Compound 2 (88 g, 200 mmol)
and triethylamine (55.8 mL, 400 mmol) were dissolved in 1 L of DCM
under inert atmosphere. The solution was cooled to 0 °C, and di-tert-
butyl dicarbonate (45.8 g, 210 mmol) was added in small portions.
The reaction mixture was slowly warmed to room temperature and
stirred for an additional 18 h. Afterward, the mixture was evaporated
under reduced pressure, and the residue was redissolved in 800 mL of
diethyl ether. The organic layer was washed twice with 500 mL of satd
sodium bicarbonate solution, dried over NaSO4, and evaporated under
reduced pressure. The crude product was recrystallized from hexane.
Compound 4 (81 g, 150 mmol) was isolated in form of a white
crystalline solid with a yield of 75%: mp = 95−98 °C; IR (film) ν
EXPERIMENTAL SECTION
■
General Methods. Unless otherwise noted, all solvents were
HPLC grade. Regarding DCM, amylene-stabilized HPLC grade was
chosen. The solid-support resin was purchased from Rapp Polymers.
Reversed-phase HPLC (RP-HPLC) was performed with 214 nm UV
detection on a C18 (Agilent Eclipse, 4.6 × 100 mm) analytical column
at 60 °C and a flow rate of 1 mL/min. Eluent: (A) water + 0.1% TFA;
(B) acetonitrile + 0.1% TFA. Strong cation-exchange HPLC (SCX-
HPLC) was performed with a 214 nm UV detection on a PL-SCX
(Agilent, 1000 Å 8 μm 150 × 4.6 mm) analytical column at 60 °C and
a flow rate of 1 mL/min. Eluent: (A) 20 mmol NaCl in H2O + 10
mmol HCl; (B) 1.5 mol of NaCl in H2O + 10 mmol of HCl were
used. The purity was determined by integration of the UV-signal with
the software ChemStation for LC from Agilent Technologies.
1
3306, 1709, 1174, 1145 cm−1; H NMR (400 MHz, CDCl3) δ 8.00
(bs, 1H), 7.39−7.33 (m, 6H), 7.23−7.16 (m, 6H), 7.13−7.08 (m, 3H),
3.38−3.29 (m, 4H), 3.21 (t, J = 5.8 Hz, 2H), 2.24 (t, J = 5.7 Hz, 2H),
1.31 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 158.2, 157.9, 157.7,
157.4, 145.7, 128.4, 127.9, 126.4, 120.1, 117.2, 114.4, 111.5, 81.0, 70.8,
Building Block Synthesis. Synthesis of N1-(2-Aminoethyl)-N2-
tritylethane-1,2-diamine (1). Diethylenetriamine (135 mL, 1.25
mol) was dissolved in 3.75 L of DCM and cooled to 0 °C.
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48.5, 45.6, 42.3, 40.8, 28.2; ESI-HRMS calcd for C30H35F3N3O3 [M +
H]+ 542.2631, found 542.2606; calcd for C30H34F3N3NaO3 [M + Na]+
564.2450, found 564.2424.
MHz, CDCl3) δ 175.6, 172.9, 157.0, 156.7, 143.8, 141.2, 132.4, 127.8,
127.0, 125.0, 119.9, 117.9, 66.7, 66.5, 47.8, 47.1, 40.1, 39.9, 39.0, 30.6,
29.5; ESI-HRMS calcd for C27H31N3NaO7 [M + Na]+ 532.2060, found
532.2067; RP-HPLC analysis, 5% to 95% MeCN in 10 min, tR = 7.8
min
Synthesis of Allyl (2-((((9H-Fluoren-9-yl)methoxy)carbonyl)-
amino)ethyl)(2(tritylamino)ethyl)carbamate (5). Compound 3
(45.6 g, 87 mmol) was dissolved in 72 mL of water and 720 mL of
methanol. Then K2CO3 (60 g, 5 equiv) was added. The suspension
was stirred for 18 h until the methanol was evaporated, and an
additional 400 mL of water, 470 mL of THF, and 9-fluorenylmethyl
chloroformate (Fmoc-Cl) (22.5 g, 27 mmol) were added. The mixture
was stirred for an additional 18 h, and then THF was evaporated and
the product extracted with diethyl ether. The organic layer was washed
twice with satd sodium bicarbonate solution, dried over Na2SO4, and
evaporated under reduced pressure. The carbamate 5 was recrystal-
lized from toluene as a white solid (45 g, 69 mmol) with a yield of
80%: mp = 118−121 °C; IR (film) ν 3347, 1686 1239, 1146 cm−1; 1H
NMR (400 MHz, CDCl3) δ 7.77 (bs, 1H), 7.75 (bs, 1H), 7.52−7.51
(m, 2H), 7.47−7.44 (m, 6H), 7.39 (t, J = 7.4 Hz, 2 H), 7.32−7.24 (m,
8H), 7.22−7.16 (m, 3H), 5.95−5.82 (m, 1H), 5.36−5.16 (m, 3H),
4.61−4.54 (m, 2H), 4.45−4.33 (m, 2H), 4.21 (t, J = 6.7 Hz, 1 H),
3.46−3.55 (m, 6H), 2.38−2.30 (m, 2H); 13C NMR (100 MHz,
CDCl3) δ 156.9, 156.5 145.8, 143.9, 141.3, 132.7, 128.5, 127.8, 127.6,
127.0, 126.3, 119.9, 117.7, 70.7, 66.6, 66.2, 48.2, 47.4, 47,2, 42.4, 40.0;
ESI-HRMS calcd for C42H42N3O4 [M + H]+ 652.3175, found
652.3196.
Synthesis of BDS: 7-(tert-Butoxycarbonyl)-1-(9H-fluoren-9-yl)-
3,11-dioxo-2-oxa-4,7,10-triazatetradecan-14-oic Acid (8). Com-
pound 6 (76.7 g, 115 mmol) was added to a mixture of 145 mL of
acetic acid and 145 mL of trifluoroethanol at 60 °C. The reaction
mixture was stirred for 60 min. Then the solution was coevaporated
with toluene. The resulting solid was dissolved in 30 mL of toluene
and precipitated with 250 mL of ice-cold diethyl ether. The white solid
was taken without any purification and dissolved in 575 mL of DCM,
followed by addition of succinic anhydride (12 g, 120 mmol). During
stirring, triethylamine (48.1 mL, 345 mmol) was added slowly to the
mixture. After 30 min, TLC monitoring (DCM/MeOH/AcOH
90:10:1) showed complete conversion. The reaction mixture was
acidified with aqueous citric acid to pH 3 and then washed with 500
mL of 5% aqueous citric acid. The crude product was dissolved in
acetone and precipitated with aqueous 1% citric acid. The highly
viscous white oil was washed twice with 1% citric acid in water. Finally,
the solid was redissolved in EtOAc and the organic layer was washed
with water once, dried over Na2SO4, and evaporated under reduced
pressure. Compound 8 (41.4 g, 79 mmol) was obtained as hygroscopic
amorphous white solid with a yield of 69%: IR (film) ν 3285, 1743,
1
1695, 1170, 1145 cm−1; H NMR (400 MHz, CDCl3) δ 7.74 (d, J =
Synthesis of tert-Butyl (2-((((9H-Fluoren-9-yl)methoxy)carbonyl)-
amino)ethyl) (2-(Tritylamino)ethyl)carbamate (6). Compound 4 (80
g, 148 mmol) was dissolved in 57 mL of water and 680 mL of
methanol. Then K2CO3 (102 g, 5 equiv) was added. The suspension
was stirred for 18 h until methanol was evaporated, and an additional
310 mL of water and 370 mL of THF and Fmoc-Cl (40 g, 155 mmol)
were added. The mixture was stirred for an additional 18 h, and then
THF was evaporated and the product was extracted with 800 mL of
diethyl ether. The organic layer was washed twice with 500 mL of satd
NaHCO3 solution, dried over Na2SO4, and evaporated under reduced
pressure. The crude product was recrystallized from hexane.
Compound 6 (91 g, 136 mmol) was obtained as a white solid with
a yield of 92%: mp = 154−156 °C; IR (film) ν 3351, 1678 1238, 1143
7.5 Hz, 2H), 7.58 (d, J = 7.4 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.29 (t,
J = 7.3 Hz, 2H), 4.48−4.28 (m, 2H), 4.18 (t, J = 6.6 Hz, 1 H), 3.44−
3.05 (m, 8H), 2.70−2.60 (m, 2H), 2.54−2.42 (m, 2H), 1.42 (bs, 9H);
13C NMR (100 MHz, CDCl3) δ 175.6, 175.5, 156.6, 143.8, 141.2,
127.7, 127.0, 125.0, 119.9, 80.7, 66.6, 48.0, 47.1, 40.5, 40.1, 39.3, 30.7,
29.8, 28,3; ESI-HRMS calcd for C28H34N3O7 [M − H]− 524.2397,
found 524.2399; RP-HPLC analysis, 5% to 95% MeCN in 10 min, tR =
8.4 min.
Solid-Phase Synthesis. All solid-phase reactions were performed
on an automated standard peptide synthesizer in 0.05 mmol scale
referring to the following general solid-phase protocols. Tentagel S
RAM resin (loading 0.23 mmol/g) was used as solid support, which
was swollen twice for 15 min in DCM before starting the initial Fmoc-
deprotection.
General Procedure. Coupling/Fmoc-Deprotection Protocol.
ADS (5 equiv, 127 mg) or BDS (5 equiv, 131 mg) and O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluor-
ophosphate (HATU) (93 mg, 4.9 equiv) were placed as
powder in the amino acid vial and placed in the peptide
synthesizer. The solids were dissolved in 2 mL of DMF with a
gentle nitrogen stream. Then 10 equiv of 1 M DIEA solution in
DMF was added. Preactivation was carried out for 3 min in the
amino acid vial before the solution was transferred to the resin.
Afterward the resin with the coupling solution was shaken
carefully for 1 h, the reaction vessel was emptied and washed
with DMF. Then the whole procedure was repeated once.
Fmoc deprotection was performed using 25% piperidine in DMF
for 5 min and checked by UV monitoring for the fluorenyl piperidine
adduct at 301 nm. This step was repeated until the deprotection was
complete.
Acetylation of the N-Terminal Side. For acetylation of the N-
terminal side 3 mL of Ac2O was placed in the amino acid vial and
transferred to the reaction vessel, which was shaken for 5 min.
Afterward the resin was washed with DMF.
Automated Alloc Cleavage. Tetrakis(triphenylphosphine)-
palladium(0) (0.1 equiv per Alloc moiety) and N,N-dimethylbarbituric
acid (5 equiv per Alloc moiety) were placed in the amino acid vial
flushed under argon. Then 4.5 mL of DCM was added to the amino
acid vial, and the solids were dissolved by a gentle nitrogen stream for
4 min. The solution was transferred to the reaction vessel, which was
shaken for 2 h. The whole procedure was repeated once before the
resin was washed 3× with DCM, 3 × 0.2 M DIEA in DMF, and 6 ×
DMF.
1
cm−1; H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 7.5 Hz, 2H), 7.58
(d, J = 7.3 Hz, 2H), 7.46 (d, J = 7.6 Hz, 6H), 7.38 (t, J = 7.4 Hz, 2 H),
7.31−7.16 (m, 8H), 7.21−7.15 (t, J = 7.2 Hz), 4.50−4.34 (m, 2H),
4.20 (t, J = 6.9 Hz, 1 H), 3.40−3.12 (m, 6H), 2.36−2.28 (m, 2H),
1.50−1.35 (m, 9H); 13C NMR (100 MHz, CDCl3) δ 156.6, 145.9,
143.9, 141.3, 128.5, 127.8, 127.6, 127.0, 126.3, 119.9, 80.1, 70.7, 66.7,
48.3, 47.3, 46,8, 42.4, 40.4, 28.4; ESI-HRMS calcd for C43H46N3O4 [M
+ H]+ 668.3488, found 668.3462.
Synthesis of ADS: 7-((Allyloxy)carbonyl)-1-(9H-fluoren-9-yl)-3,11-
dioxo-2-oxa-4,7,10-triazatetradecan-14-oic Acid (7). Compound 5
(43 g, 66 mmol) was dissolved in 627 mL of DCM and triethylsilane
(31.6 mL, 198 mmol). After addition of 33 mL of TFA (5 vol%
regarding the reaction mixture) at 0 °C, the mixture was allowed to
reach rt. The reaction was complete after 30 min according to TLC
monitoring (2:1 Hex/EtoAc). Then the reaction mixture was
coevaporated with toluene. The resulting solid was dissolved in 30
mL of toluene and precipitated with 200 mL of diethyl ether. The
white solid was dissolved with succinic anhydride (6.9 g, 69.3 mmol)
in 660 mL of DCM, and triethylamine (27.6 mL, 198 mmol) was
added carefully to the mixture. After 30 min, complete conversion was
monitored on TLC (DCM/MeOH/AcOH 90:10:1). The reaction
mixture was acidified with aqueous citric acid to pH 3 and washed with
500 mL of 5% aqueous citric acid. The organic layer was dried over
Na2SO4 and evaporated under reduced pressure. The crude product
was recrystallized from a mixture of toluene/ethanol 20:1. Compound
7 (25.6 g, 50 mmol) was obtained as a white crystalline solid with a
yield of 76%: mp = 81−84 °C; IR (film) ν 3320, 1716, 1678, 1645,
1237, 1142 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 7.5 Hz,
2H), 7.57 (d, J = 7.4 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2 H), 7.29 (t, J = 7.4
Hz, 2 H), 5.91−5.81 (m, 1H), 5.28−5.15 (m, 2H), 4.56−4.45 (m,
2H), 4.41−4.31 (m, 2H), 4.18 (t, J = 6.8 Hz, 1 H), 3.45−3.15 (m,
8H), 2.65 (t, J = 6.2 Hz, 2H), 2.49−2.40 (m, 2H); 13C NMR (100
4232
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Article
Side-Chain Elongation. The side-chain elongation was performed
with the previously described protocol for coupling and deprotection,
but 5 equiv of BDS, 4.9 equiv of HATU, and 10 equiv of 1 M DIEA
per side chain were used. The solids were dissolved in 4 mL of DMF.
Final Cleavage from Solid Support. The final cleavage was
performed by adding the cleavage cocktail (95% TFA, 2.5%
triisopropylsilane, and 2.5% water 1 mL/50 mg resin) to the resin
and allowing it to react for 70 min. The cleavage solution was filtered
and purged into ice-cold diethyl ether. The precipitate was isolated and
washed twice with diethyl ether. The residue was dissolved in water
and lyophilized overnight, giving the final product and the
corresponding yield.
AcHN-ADS-ADS-ADS-ADS-ADS-CONH2 (9). Compound 9 was
cleaved from the resin on an analytical scale. The resin-bound product
was used for further reactions: MALDI-TOF calcd for C62H101N16O21
[M + H]+ 1405.733 (monoisotopic), found 1405.711; calcd for
C62H100N16NaO21 [M + Na]+ 1427.72 (monoisotopic), found
1427.70; calcd for C62H100KN16O21 [M + K]+ 1443.69 (mono-
isotopic), found 1443.68; RP-HPLC analysis 5% to 50% MeCN in 60
min, tR = 29.2 min.
AcNH-[DS(BDS-Fmoc)-BDS]4-CONH2 (16). Compound 16 was
cleaved from the resin on an analytical scale. The resin-bound product
was used for further reactions: MALDI-TOF calcd for C158H226N37O33
[M + H]+ 3169.71 (monoisotopic), found 3169.71; calcd for
C158H225N37NaO33 [M + Na]+ 3191.70 (monoisotopic), found
3196.69; calcd for C158H225KN37O33 [M + K]+ 3207.67 (mono-
isotopic), found 3207.66; RP-LC/MS analysis 5% to 50% MeCN in 60
min, tR = 43.4 min; calcd for C158H227N37O33 [M + 2H]2+ 1586.4
(mean), found 1586.3; calcd for C158H228N37O33 [M + 3H]3+ 1057.9
(mean), found 1058.1; calcd for C158H229N37O33 [M + 4H]4+ 793.7
(mean); found 794.0; calcd for C158H230N37O33 [M + 5H]5+ 635.1
(mean); found 635.5.
AcNH-[DS(BDS-NH2)-BDS]4-CONH2 (17). Compound 17 (77 mg,
1
0.034 mmol) was obtained with a yield of 68%. H NMR (400 MHz,
D2O) δ 3.70−3.20 (m, diamine unit, 96H), 2.74−2.50 (m, diacid unit,
48H), 2.02 (s, acetyl, 3H); MALDI-TOF calcd for C98H186N37O25 [M
+ H]+ 2281.44 (monoisotopic); found 2281.47; calcd for
C98H185N37NaO25 [M + Na]+, 2303.42 (monoisotopic); found
2303.43; calcd for C98H185KN37O25 [M + K]+, 2319.40 (mono-
isotopic), found 2319.41.
AcNH-[BDS-DS(BDS-Fmoc)-BDS]3-CONH2 (18). Compound 18 was
cleaved from the resin on an analytical scale. The resin-bound product
was used for further reactions: MALDI-TOF calcd for C143H216N37O31
[M + H]+ 2947.65 (monoisotopic), found 2947.73; calcd for
C143H215N37NaO31 [M + Na]+ 2969.63 (monoisotopic), found
2281.70; calcd for C143H215KN37O31 [M + K]+ 2985.60 (mono-
isotopic), found 2985.68; RP-LC/MS analysis 5% to 50% MeCN in 60
min, tR = 36.5 min; calcd for C143H218N37O31 [M + 3H]3+ 983.8
(mean), found 984.0; calcd for C143H219N37O31 [M + 4H]4+ 738.1
(mean), found 738.4.
AcNH-(ADS-BDS)4-CONH2 (10). Compound 10 was cleaved from
the resin on an analytical scale. The resin-bound product was used for
further reactions: MALDI-TOF calcd for C82H142N25O25 [M + H]+
1877.06 (monoisotopic), found 1877.08; calcd for C82H141N25NaO25
[M + Na]+ 1899.04 (monoisotopic), found 1899.06; calcd for
C82H141KN25O25 [M + K]+ 1915.02 (monoisotopic), found 1915.03;
RP-HPLC analysis 5% to 50% MeCN in 60 min, tR = 18.8 min.
AcNH-(BDS-ADS-BDS)3-CONH2 (11). Compound 11 was cleaved
from the resin on an analytical scale. The resin-bound product was
used for further reactions: MALDI-TOF calcd for C86H153N28O25 [M
+ H]+ 1978.156 (monoisotopic), found 1978.131; calcd for
C86H152N28NaO25 [M + Na]+ 2000.14 (monoisotopic), found
2000.12; calcd for C86H152KN28O25 [M + K]+ 2016.11 (mono-
isotopic), found 2016.09; RP-HPLC analysis 5% to 50% MeCN in 60
min, tR = 12.9 min.
AcNH-(BDS-ADS-BDS)4-CONH2 (12). Compound 12 was cleaved
from the resin on an analytical scale. The resin-bound product was
used for further reactions. MALDI-TOF calcd for C114H202N37O33 [M
+ H]+ 2617.53 (monoisotopic), found 2617.53; calcd for
C114H201N37NaO33 [M + Na]+ 2639.51 (monoisotopic), found
2639.51; calcd for C114H201KN37O33 [M + K]+ 2655.48 (mono-
isotopic), found 2655.49; RP-HPLC analysis 5% to 30% MeCN in 60
min, tR = 22.9 min.
AcNH-(BDS-ADS-BDS)5-CONH2 (13). Compound 13 was cleaved
from the resin on an analytical scale. The resin-bound product was
used for further reactions: MALDI-TOF calcd for C142H251N46O41 [M
+ H]+ 3256.90 (monoisotopic), found 3256.92; calcd for
C142H250N46NaO41 [M + Na]+ 3278.88 (monoisotopic), found
3278.84; calcd for C142H250KN46O41 [M + K]+ 3294.85 (mono-
isotopic), found 3294.82; RP-HPLC analysis 5% to 30% MeCN in 60
min, tR = 24.6 min.
AcNH-[DS(BDS-Fmoc)]5-CONH2 (14). Compound 14 was cleaved
from the resin on an analytical scale. The resin-bound product was
used for further reactions: MALDI-TOF calcd for C157H205N31NaO31
[M + Na]+ 3043.53 (monoisotopic), found 3043.51; calcd for
C157H205KN31O31 [M + K]+ 3059.51 (monoisotopic), 3059.50; RP-
LC/MS analysis 5% to 50% MeCN in 60 min, tR = 53.5 min, calcd for
C157H207N31O31 [M + 2H]2+ 1512.3 (mean), found 1512.7; calcd for
C157H208N31O31 [M + 3H]3+ 1008.5 (mean), found 1008.7; calcd for
C157H209N31O31 [M + 4H]4+ 756.6 (mean), found 756.6; calcd for
C157H210N31O31 [M + 5H]5+ 605.5 (mean), found 605.7.
AcNH-[BDS-DS(BDS-NH2)-BDS]3-CONH2 (19). Compound 19 (90
1
mg, 0.040 mmol) was obtained with a yield of 79%: H NMR (400
MHz, D2O) δ 3.70−3.20 (m, diamine unit, 96H), 2.74−2.51 (m,
diacid unit, 48H), 2.01 (s, acetyl, 3H); MALDI-TOF calcd for
C98H186N37O25 [M + H]+ 2281.44 (monoisotopic), found 2281.52;
calcd for C98H185N37NaO25 [M + Na]+ 2303.42 (monoisotopic),
found 2303.49.
AcNH-[BDS-DS(BDS-Fmoc)-BDS]4-CONH2 (20). Compound 20 was
cleaved from the resin on an analytical scale. The resin-bound product
was used for further reactions: MALDI-TOF calcd for
C190H285N49NaO41 [M + Na]+ 3932.16 (monoisotopic), found
3932.14; RP-LC/MS analysis, 5% to 50% MeCN in 60 min, tR
=
39.3 min; calcd for C190H288N49O41 [M + 3H]3+ 1304.9 (mean), found
1305.1; calcd for C190H289N49O41 [M + 4H]4+ 978.9 (mean), found
978.3; calcd for C190H290N49O41 [M + 5H]5+ 783.3 (mean), found
783.7; calcd for C190H291N49O41 [M + 6H]6+ 652.9 (mean), found
653.2.
AcNH-[BDS-DS(BDS-NH2)-BDS]4-CONH2 (21). Compound 21 (107
1
mg, 0.036 mmol) was obtained with a yield of 71%: H NMR (400
MHz, D2O) δ 3.71−3.22 (m, diamine unit, 128H), 2.75−2.52 (m,
diacid unit, 64H), 2.03 (s, acetyl, 3H); MALDI-TOF calcd for
C130H246N49O33 [M + H]+, 3021.91 (monoisotopic), found 3021.87;
calcd for C130H245N49NaO33 [M + Na]+ 3043.89 (monoisotopic),
found 3043.81.
AcNH-[BDS-DS(BDS-Fmoc)-BDS]5-CONH2 (22). Compound 22 was
cleaved from the resin on an analytical scale. The resin-bound product
was used for further reactions: MALDI-TOF calcd for C237H356N61O51
[M + H]+, 4872.51 (monoisotopic), found 4872.28; calcd for
C237H355N61NaO51 [M + Na]+ 4894.70 (monoisotopic), found
4894.28; RP-LC/MS analysis 5% to 50% MeCN in 60 min, tR
=
42.3 min; calcd for C237H359N61O51 [M + 4H]4+ 1219.7 (mean), found
1219.6; calcd for C237H360N61O51 [M + 5H]5+ 975.9 (mean), found
975.8; calcd for C237H361N61O51 [M + 6H]6+ 813.5 (mean), found
813.4; calcd for C237H362N61O51 [M + 7H]7+ 697.4 (mean), found
697.4; calcd for C237H363N61O51 [M + 8H]8+ 610.3 (mean), found
610.3.
AcNH-[DS(BDS-NH2)]5-CONH2 (15). Compound 15 (71 mg, 0.037
mmol) was obtained with a yield of 74%: 1H NMR (400 MHz, D2O) δ
3.71−3.29 (m, diamine unit, 80H), 2.75−2.49 (m, diacid unit, 40H),
1.98 (d, J = 13.7 Hz, acetyl, 3H); MALDI-TOF calcd for
C82H156N31O21 [M + H]+ 1911.21 (monoisotopic), found 1911.15;
calcd for C82H155N31NaO21 [M + Na]+ 1933.19 (monoisotopic),
found 1933.09; SCX-HPLC analysis 20 mmol to 1.5 mol NaCl in 30
min, tR = 20.2 min.
AcNH-[BDS-DS(BDS-NH2)-BDS]5-CONH2 (23). Compound 23 (124
1
mg, 0.033 mmol) was obtained with a yield of 66%: H NMR (400
MHz, D2O) δ 3.71−3.21 (m, diamine unit, 160H), 2.75−2.53 (m,
diacid unit, 80H), 2.02 (s, acetyl, 3H); MALDI-TOF calcd for
4233
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The Journal of Organic Chemistry
Article
C162H305N61NaO41 [M + Na]+ 3784.36 (monoisotopic), found
3784.32.
AcNH-[DS(BDS-BDS-Fmoc)-BDS]4-CONH2 (24). Compound 24 was
cleaved from the resin on an analytical scale. The resin-bound product
was used for further reactions: MALDI-TOF calcd for
C190H285N49NaO41 [M + Na]+ 3932.16 (monoisotopic), found
(13) Mosca, S.; Wojcik, F.; Hartmann, L. Macromol. Rapid Commun.
2011, 32, 197−202.
(14) Schaffert, D.; Badgujar, N.; Wagner, E. Org. Lett. 2011, 13,
1586−1589.
(15) Hartmann, L.; Borner, H. G. Adv. Mater. 2009, 21, 3425−3431.
(16) Hartmann, L. Macromol. Chem. Phys. 2011, 212, 8−13.
(17) Krishna, T. R.; Jayaraman, N. J. Org. Chem. 2003, 68, 9694−
9704.
3932.07; RP-LC/MS analysis 5% to 50% MeCN in 60 min, tR
=
40.7 min; calcd for C190H288N49O41 [M + 3H]3+ 1304.9 (mean), found
1305.0; calcd for C190H289N49O41 [M + 4H]4+ 978.9 (mean), found
978.3; calcd for C190H290N49O41 [M + 5H]5+ 783.3 (mean), found
783.8; calcd for C190H291N49O41 [M + 6H]6+ 652.9 (mean), found
653.3.
(18) Fischer, D.; Bieber, T.; Li, Y. X.; Elsasser, H. P.; Kissel, T.
Pharm. Res. 1999, 16, 1273−1279.
(19) Liu, M.; Chen, J.; Cheng, Y.-P.; Xue, Y.-N.; Zhuo, R.-X.; Huang,
S.-W. Macromol. Biosci. 2010, 10, 384−392.
AcNH-[DS(BDS-BDS-NH2)-BDS]4-CONH2 (25). Compound 25 (110
(20) Caminade, A. M.; Turrin, C. O.; Majoral, J. P. Chem.Eur. J.
1
mg, 0.037 mmol) was obtained with a yield of 73%: H NMR (400
2008, 14, 7422−7432.
MHz, D2O) δ 3.71−3.24 (m, diamine unit, 128H), 2.75−2.50 (m,
diacid unit, 64H), 2.02 (s, acetyl, 3H); MALDI-TOF calcd for
C130H246N49O33 [M + H]+ 3021.91 (monoisotopic), found 3021.91;
calcd for C130H245N49NaO33 [M + Na]+ 3043.89 (monoisotopic),
found 3043.86.
(21) Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.;
Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 7297−7301.
(22) Neu, M.; Fischer, D.; Kissel, T. J. Gene. Med. 2005, 7, 992−1009.
(23) Isidro-Llobet, A.; Alvarez, M.; Albericio, F. Chem. Rev. 2009,
109, 2455−2504.
ASSOCIATED CONTENT
(24) Schelhaas, M.; Waldmann, H. Angew. Chem., Int. Ed. 1996, 35,
2056−2083.
■
S
* Supporting Information
(25) Dangles, O.; Guibe, F.; Balavoine, G.; Lavielle, S.; Marquet, A. J.
Org. Chem. 1987, 52, 4984−4993.
More experimental details, copies of the H and 13C NMR
1
spectra, RP-HPLC analysis, SCX-HPLC analysis, LC/MS
analysis, and mass spectra of all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org
(26) Verheijen, J. C.; Deiman, B. A. L. M.; Yeheskiely, E.; van der
Marel, G. A.; van Boom, J. H. Angew. Chem., Int. Ed. 2000, 39, 369−
372.
(27) Xu, D.; Prasad, K.; Repic, O.; Blacklock, T. J. Tetrahedron Lett.
1995, 36, 7357−7360.
AUTHOR INFORMATION
Corresponding Author
*E-mail: laura.hartmann@mpikg.mpg.de.
Notes
■
(28) Hackenberger, C. P. R; Schwarzer, D. Angew. Chem., Int. Ed.
2008, 47, 10030−10074.
(29) Wuts, P. G. M.; Greene, T. W. Greene’s Protective Groups in
Organic Synthesis, 4th ed.; John Wiley & Sons: New York, 2007.
(30) Albericio, F.; Bofill, J. M.; El-Faham, A.; Kates, S. A. J. Org.
Chem. 1998, 63, 9678−9683.
(31) Wohr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.
C.; Mutter, M. J. Am. Chem. Soc. 1996, 118, 9218−9227.
(32) Kates, S. A.; Daniels, S. B.; Albericio, F. Anal. Biochem. 1993,
212, 303−310.
(33) Zorn, C.; Gnad, F.; Salmen, S.; Herpin, T.; Reiser, O.
Tetrahedron Lett. 2001, 42, 7049−7053.
(34) Unverzagt, C.; Kunz, H. Bioorg. Med. Chem. 1994, 2, 1189−
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Dr. P. H. Seeberger for support and helpful
discussions as well as Dr. Nicole Snyder and Dr. Daniel Varon
Silva. We also thank Dr. Alexander O’Brien, Dr. Daniel
Kolarich, Christopher Martin, Olaf Niemeyer, Daniela Ponader,
Eva Settels, and the Mass Spectrometry Core Facility at the
Freie Universitat Berlin for technical support. Financial support
̈
was granted by the German Research Foundation (DFG)
through the Emmy Noether program (HA5950/1-1) and the
Max Planck Society.
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