From peptides to their alternating ester-urea analogues: Synthesis and influence of hydrogen bonding motif and stereochemistry on aggregation

Article abstract of DOI:10.1021/jo902249w

(Chemical Equation Presented) Peptide-mimicking scaffolds with an incorporated ester-urea motif, replacing two adjacent amide residues, were synthesized and their aggregation behavior was studied in dependence of hydrogen bonding sites as well as backbone stereochemistry. Two oligomer series containing either 50% or 100% ester-urea units and either all-(L) or (D)-alt-(L) backbone configuration were prepared via ester and amide couplings, using a divergent/convergent exponential growth strategy. Their aggregation behavior in organic solution was investigated by means of concentration-dependent NMR spectroscopy and compared to the parent peptide series. Interestingly, the naturally occurring peptide scaffold exhibits the largest tendency to associate in combination with the strongest difference in aggregation behavior between all-(L) and (D)-alt-(L) backbone stereochemistry. With increasing incorporation of the ester-urea motif the aggregation strength decreases and become much less dependent on the backbone configuration. The obtained structure-aggregation relationships reveal the importance of the commensurability and multivalency of hydrogen bonding sites as well as conformational restriction for peptide association and should hence aid the design of peptide mimics, such as β -sheet breakers or gelators. 2009 American Chemical Society.

Full text of DOI:10.1021/jo902249w

pubs.acs.org/joc
From Peptides to Their Alternating Ester-Urea Analogues: Synthesis and
Influence of Hydrogen Bonding Motif and Stereochemistry on
Aggregation
Sebastian Hartwig, Jutta Schwarz, and Stefan Hecht*
€
Department of Chemistry, Humboldt-Universitat zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany
sh@chemie.hu-berlin.de
Received October 29, 2009
Peptide-mimicking scaffolds with an incorporated ester-urea motif, replacing two adjacent amide
residues, were synthesized and their aggregation behavior was studied in dependence of hydrogen
bonding sites as well as backbone stereochemistry. Two oligomer series containing either 50% or
100% ester-urea units and either all-(L) or (D)-alt-(L) backbone configuration were prepared via ester
and amide couplings, using a divergent/convergent exponential growth strategy. Their aggregation
behavior in organic solution was investigated by means of concentration-dependent NMR spectro-
scopy and compared to the parent peptide series. Interestingly, the naturally occurring peptide
scaffold exhibits the largest tendency to associate in combination with the strongest difference in
aggregation behavior between all-(L) and (D)-alt-(L) backbone stereochemistry. With increasing
incorporation of the ester-urea motif the aggregation strength decreases and become much less
dependent on the backbone configuration. The obtained structure-aggregation relationships reveal
the importance of the commensurability and multivalency of hydrogen bonding sites as well as
conformational restriction for peptide association and should hence aid the design of peptide mimics,
such as β-sheet breakers or gelators.
Introduction
peptides is an important field of research,2 but also the
integration of other functionalities into the polyamide back-
bone of a peptide is promising to gain detailed insight into
structure-function relationships, subsequently leading to
improved properties.3
One possibility of varying the backbone of a peptide is the
replacement of amides with esters. For incorporation of an
ester as an isostere into the sequence, an R-amino acid needs
to be exchanged by an R-hydroxy acid. This replacement
The generation and elucidation of peptide structures is
crucial for both understanding biological processes and
developing biologically active materials suitable for pharma-
ceutical and medical applications.1 With this in mind, it
becomes apparent that not only the development of new
(1) (a) Marx, V. Chem. Eng. News 2005, 83, 17. (b) Gentilucci, L.;
Tolomelli, A.; Squassabia, F. Curr. Med. Chem. 2006, 13, 2449.
(2) (a) Peptide-Based Drug Design: Methods and Protocols; Otvos, L., Ed;
Springer, Berlin, Germany, 2008; (b) Caliceti, P.; Veronese, F. M. Adv. Drug
Delivery Rev. 2003, 55, 1261. (c) Trends and Future Perspectives in Peptide and
Protein Drug Delivery; Lee, V., Ed.; Taylor & Francis: New York, 1995.
(3) (a) Advances in Amino Acid Mimetics and Peptidomimetics; Abell,
A. D., Ed.; JAI Press: New York, 1999; Vol. 2. (b) Pseudo-Peptides in Drug
Discovery; Nielsen, P. E., Ed.; Wiley-VCH: Weinheim, Germany, 2004.
772 J. Org. Chem. 2010, 75, 772–782
Published on Web 12/29/2009
DOI: 10.1021/jo902249w
r
2009 American Chemical Society

Hartwig et al.
JOCArticle
replaced by an “ester-urea” moiety (see Figure 1, bottom
right). This urea incorporation elongates6 the backbone by
one further atom and adds one hydrogen bond acceptor. The
ester-urea structure has the same main chain direction as the
original peptide and also the same number of hydrogen bond
donor and acceptor sites. The location and the direction of
these hydrogen bond donors and acceptors in the backbone
differ significantly from those in peptides. The incorporation
of an ester interrupts the highly ordered hydrogen bonding
pattern of the peptide backbone, whereas the urea unit is
engaged in stronger hydrogen bonding interactions and
hence may be able to compensate.7,8 This linkage variation
results in a very interesting type of hydrogen bonding pattern
and thereby in a potentially novel secondary (and higher)
structure(s). Consequent replacement of every amide in the
main chain affords an ester-alt-urea backbone, which con-
stitutes an interesting and novel class of compounds with
potentially advantageous properties, such as improved bio-
degradability and bioavailability. In addition to linkage
chemistry the incorporation of (D)-configured amino acid
building blocks presents another means to largely affect
peptide secondary structures, e.g., in the alternating (D)-
alt-(L) Gramicidin channels,9,10 thereby causing significantly
different pharmacological characteristics.
FIGURE 1. Peptide backbone modifications leading to depsipep-
tides, “ester-amides”, and novel structural motifs, such as the
investigated “ester-ureas”.
This simple structural gedankenexperiment encouraged us
to prepare and investigate oligomers with varying degree of
ester-urea incorporation and different stereochemistry, i.e.
all-(L) and (L)-alt-(D).11 Here, we present the synthesis of
these two new peptide mimicking backbones and report
some initial investigations of the association behavior of
their short oligomers.
maintains the direction of the peptide sequence (i.e., from
N- to C-terminus) but eliminates one hydrogen bond donor
site (see Figure 1 on top). This field of depsipeptides has been
thoroughly investigated over the past decades,4 in particular
with regard to folding behavior.
Another interesting possibility to exchange an amide by an
ester is the replacement of an R-amino acid in the sequence by
the corresponding amino alcohol (see Figure 1, bottom left).
This exchange introduces an ester under elongation5 of the
backbone by one atom and eliminates one hydrogen bond
acceptor site. This amide-ester exchange goes in hand with a
reversal of the main chain direction in the ester-amide back-
bone.
Results and Discussion
General Design Considerations. To investigate the influence
of the connectivity (isostere incorporation) and stereochemis-
try, three different series ranging from peptides to completely
alternating ester-urea pseudopeptides each composed of both
One option to maintain the direction of the peptide is the
further incorporation of a urea moiety into the backbone,5 so
that altogether two adjacent amino acids in the peptide are
(6) Elongation refers to the side chain repetition, i.e., the number of atoms
in between neighboring monomer repeat units.
(7) Ureas have been used to facilitate aggregation and subsequent crystal-
lization or gelation: (a) Desiraju, G. R., Crystal Engineering: The Design of
Organic Solids; Elsevier: Amsterdam, The Netherlands, 1989; (b) Jadzyn, J.;
Stockhausen, M.; Zywucki, B. J. Phys. Chem. 1987, 91, 754. (c) Chang, Y.-L.;
West, M.-A.; Fowler, F. W.; Lauher, J. W. J. Am. Chem. Soc. 1993, 115, 5991.
(d) van Esch, J. H.; Kellogg, R. M.; Feringa, B. L. Tetrahedron Lett. 1997, 38,
281. (e) van Esch, J. H.; Schoonbeek, F.; de Loos, M.; Kooijman, H.; Spek, A. L.;
Kellogg, R. M.; Feringa, B. L. Chem.;Eur. J. 1999, 5, 937. (f) van Esch, J. H.;
Feringa, B. L. Angew. Chem., Int. Ed. 2000, 39, 2263. (g) Schoonbeek, F. S.; van
Esch, J. H.; Hulst, R.; Kellogg, R. M.; Feringa, B. L. Chem.;Eur. J. 2000, 6,
2633. (h) de Loos, M.; Ligtenbarg, A. G. J.; van Esch, J. H.; Kooijman, H.; Spek,
A. L.; Hage, R.; Kellogg, R. M.; Feringa, B. L. Eur. J. Org. Chem. 2000, 3675.
For a theoretical treatment of urea aggregation, see: (i) Stumpe, M. C.; Grubm€uller,
H. J. Phys. Chem. B 2002, 111, 6220.
(4) For selected examples, see: (a) Shemyakin, M. M. Angew. Chem. 1960,
72, 342. (b) Schwyzer, R.; Carrion, J. P. Helv. Chim. Acta 1960, 43, 2101.
(c) Schulz, H. Chem. Ber. 1966, 99, 3425. (d) Shemyakin, M. M.; Shchukina,
L. A.; Vinogradova, E. I.; Ravdel, G. A.; Ovchinnikov, Yu. A. Experientia
1966, 22, 535. (e) Ingwall, R. T.; Goodman, M. Macromolecules 1974, 7, 598.
(f) Becktel, W.; Wouters, G.; Simmons, D. M.; Goodman, M. Macromole-
cules 1985, 18, 630. (g) Goodman, M. Biopolymers 1985, 24, 137. (h) Gallo,
E. A.; Gellman, S. H. J. Am. Chem. Soc. 1993, 115, 9774. (i) Katakai, R.;
Kobayashi, K.; Yonezawa, N.; Yoshida, M. Biopolymers 1996, 38, 285.
(j) Karle, I. L.; Das, C.; Balaram, P. Biopolymers 2001, 59, 276. (k) Jude, A.
R.; Providence, L. L.; Schmutzer, S. E.; Shobana, S.; Greathouse, D. V.;
Andersen, O. S.; Koeppe, R. E. Biochemistry 2001, 40, 1460. (l) Oku, H.;
Yamada, K.; Katakai, R. Biopolymers 2008, 89, 270.
(5) For examples of urea incorporation into peptides, see: (a) Rink, H.
Tetrahedron Lett. 1987, 28, 3787. (b) Burgess, K.; Linthicum, D. S.; Shin, H.
Angew. Chem., Int. Ed. 1995, 34, 907. (c) Kim, J. M.; Bi, Y.; Paikoff, S.;
Schultz, P. G. Tetrahedron Lett. 1996, 37, 5305. (d) Burgess, K.; Ibarzo, J.;
Linthicum, D. S.; Russell, D. H.; Shin, H.; Shitang-Koon, A.; Totani, R.;
Zhang, A. J. J. Am. Chem. Soc. 1997, 119, 1556. (e) Boeijen, A.; Liskamp,
R. M. J. Eur. J. Org. Chem. 1999, 2127. (f) Guichard, G.; Semetey, V.;
Rodriguez, M.; Briand, J.-P. Tetrahedron Lett. 2000, 41, 1553. (g) Semetey,
V.; Rognan, D.; Hemmerlin, C.; Graff, R.; Briand, J.-P.; Marraud, M.;
Guichard, G. Angew. Chem., Int. Ed. 2002, 85, 1893. (h) Hemmerlin, C.;
Marraud, M.; Rognan, D.; Graff, R.; Semetey, V.; Briand, J.-P.; Guichard,
G. Helv. Chim. Acta 2002, 85, 3692. (i) Sureshbabu, V. V.; Patil, B. S.;
Venkataramanarao, R. J. Org. Chem. 2006, 71, 7697.
(8) For reviews on small molecule gelators based on amides and ureas,
see: (a) Estroff, L. A.; Hamilton, A. D. Chem. Rev. 2004, 104, 1201.
€
(b) Fages, F.; Vogtle, F.; Zinic, M. Top. Curr. Chem. 2005, 256, 77.
(9) (a) Wallace, B. A.; Ravikumar, K. Science 1988, 241, 182. (b) Langs,
D. A. Science 1988, 241, 188. (c) Hladky, S. B.; Haydon, D. A. Nature 1970,
5231, 451.
(10) For artificial (D)-alt-(L) peptides, see for example: (a) Benedetti, E.;
Di Blasio, B.; Pedone, C.; Lorenzi, G. P.; Tomasic, L.; Gramlich, V. Nature
1979, 282, 630. (b) Lorenzi, G. P.; Tomasic, L. Makromol. Chem. 1988, 189,
207. (c) Di Blasio, B.; Benedetti, E.; Pavone, V.; Pedone, C.; Spiniello, O.;
Lorenzi, G. P. Biopolymers 1989, 28, 193.
(11) For related recent investigations, see: (a) Hartwig, S.; Hecht, S.
Macromolecules. DOI: 10.1021/ma902018w. Published Online: Oct 30, 2009.
(b) Hartwig, S.; Nguyen, M. M.; Hecht, S. Polym. Chem. DOI:10.1039/
B9PY00217K. Published Online.
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FIGURE 3. Key building block for pseudopeptide synthesis.
SCHEME 1. Synthesis of the Key Building Blocks 3a/3b
FIGURE 2. Two adjacent amide bonds in the peptide backbone
(highlighted in yellow) are successively replaced by an ester-alt-urea
moiety (ester highlighted in blue, urea highlighted in red).
all-(L)- as well as (D)-alt-(L)-configured amino acid building
blocks (Figure 2) were targeted.
procedure, as experienced in the attempted cleavage of the
methyl ester protected leucine ureas.12
Oligo-(L)- and oligo-(D)-alt-(L)-peptides having an amide
content of 100% (Figure 2, top) serve as reference com-
pounds. As a first structural variation, half of the amide
bonds of the peptide backbone can be replaced by an ester-
alt-urea moiety (Figure 2, middle). Complete replacement of
the amide bonds leads to alternating ester-urea pseudopep-
tides (Figure 2, bottom). Note that incorporation of the
ester-urea motif allows for variation of the stereochemistry,
leading to all-(L)- as well as (D)-alt-(L)-structures in the latter
two cases. Interestingly, such trivial structural variation
leads to two new backbone primary structures, which, to
the best of our knowledge, have not been reported thus far.
To restrict structural variations to connectivity and stereo-
chemistry only, strands based solely on leucine and leucinole
were investigated, as leucine carries no functionality in
the side chain, therefore facilitating synthesis. Furthermore,
we limit the discussion to the tetramer series (n = 1).
Synthesis. Synthesis of the Key Building Block. The synth-
esis of the different targeted series of compounds with
variable stereochemistry (Figure 2) requires a straight-
forward strategy based on coupling of common fragments.
The native peptides are readily accessible via a divergent/
convergent synthesis. The 50% and 0% amide containing
pseudopeptides can be derived from a common key building
block, largely minimizing the synthetic effort (Figure 3).
This key building block can readily be synthesized by
reaction of (L)-Leu-Bn and (D)-Leu-Bn, respectively, with 1,10-
carbonyldiimidazole (CDI) and subsequent coupling with
(L)-leucinole, to give both the all-(L)- as well as the (L)-alt-(D)-
diastereomeres 3a and 3b in good yields (Scheme 1).11 The
benzyl ester protecting group can quantitatively be cleaved
by hydrogenolysis with use of Pd/C under a hydrogen atmo-
sphere. Alternative cleavage conditions involving saponifi-
cation led to undesired, quantitative cyclization to the
corresponding hydantoin during the acidic aqueous workup
Synthesis of the Ester-alt-Urea Pseudopeptide Series. With
the key building blocks 3a and 3b in hand, the ester-alt-urea
tetramers can readily be synthesized via divergent/conver-
gent synthesis. One requirement for divergent/convergent
synthesis is the protection of both reactive chain ends with
orthogonal protecting groups, in order to prevent undesired
polymerization and to enable selective activation of each
terminus. While a benzyl ester served to protect the car-
boxylic acid, the alcohol functionality was protected with the
tert-butyldiphenylsilyl (TBDPS) group, allowing for orthogonal
deprotection by hydrogenolysis and fluoride, respectively.
The TBDPS-group showed superior hydrolytic stability as
compared to both triisopropylsilyl (TIPS) as well as tert-
butyldimethylsilyl (TBDMS).12
Starting from 3a/3b, a TBDPS incorporation followed by
hydrogenolysis led to 5a/5b, which were then coupled to 3a/
3b to afford the fully protected tetramers 6a/6b (Scheme 2).
An esterification protocol, involving 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDC) as coup-
ling reagent and 4-dimethylaminopyridine (DMAP) as the
catalyst (vide infra),13 was employed. Finally, the TBDPS-
protecting group was removed with HF in acetonitrile to
yield the desired ureas 7a/7b in high yields after column
chromatography.
Synthesis of the 50% Amide Containing Pseudopeptide
Series. The synthesis of the oligopseudopeptides with 50%
amide content was initiated by the formation of an ester
bond between key building blocks 3a/3b and Boc-protected
leucine (Scheme 3). Since first esterification attempts suf-
fered from poor yields and difficult purifications, the reac-
tion was screened extensively in order to elaborate the best
(12) See the Supporting Information.
€
€
(13) (a) Hofle, G.; Steglich, W.; Vorbruggen, H. Angew. Chem., Int. Ed.
Engl. 1978, 17, 569. (b) Scriven, E. F. V. Chem. Soc. Rev. 1983, 12, 129.
774 J. Org. Chem. Vol. 75, No. 3, 2010

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SCHEME 2. Synthesis of the all-(L)- and (L)-alt-(D)-Tetramers 7a/7b via a Divergent/Convergent Synthesis Approach, Starting from the
Key Building Blocks 3a and 3b, Respectively
turned out that DMAP13 and 4-dimethylaminopyridinium
SCHEME 3. Synthesis of the 50% Amide Containing all-(L)- and
the (D)-alt-(L)-Tetrapseudopeptides 10a and 10b, Respectively,
Starting from the Key Building Blocks 3a and 3b
tosylate (DPTS)14 gave much better results, quite independently
from the coupling reagent. Furthermore, the typically per-
formed aqueous workup procedure was replaced by an
alternative method involving stirring the crude reaction
mixture over silica gel immediately followed by chromato-
graphy. On the basis of this optimized protocol, the reaction
was performed on gram scales and afforded the resulting
products 8a and 8b in quantitative yields and in high purity.
Subsequently, the trimers 8a and 8b were deprotected at
their C-termini via hydrogenolysis to yield the desired pro-
ducts 9a and 9b in quantitative yields after purification via
silica column chromatography. Subsequent coupling with
C-protected leucine 1a gave the all-(L)- and (L)-alt-(D)-ester-
alt-urea-tetramers 10a and 10b in high yields and purities.
In principle, the desired tetramers 10a/10b could also be
obtained by extending 3a/3b at the C-terminus first and
esterification of the alcohol in the last step; however, this
“amide-first” route was not successful.3 Note that the ortho-
gonally protected tetramers 10a and 10b enabled further
growth of the pseudopeptides via a divergent/convergent
synthesis approach up to the stages of octamers as well as
hexadecamers.12
Synthesis of the Peptide Series. The reference (D)-alt-(L)-
and all-(L)-leucine tetramers were synthesized via a diver-
gent/convergent synthesis, involving the orthogonally Boc/
Bn-ester protected dileucine building block, which is readily
available by coupling either (L)- or (D)-N-Boc-protected
leucine with (L)-C-Bn-protected leucine.12 Note that solution
synthesis instead of well-established solid phase supported
peptide synthesis was used with the option to prepare the
desired peptides on multigram scales.
All of the compounds synthesized constitute rather small
coupling conditions and workup procedures.12 Summarizing
the results from this screening, it is important to point out the
role of the coupling catalysts for esterification. 1-Hydroxy-
benzotriazole (HOBT), which is one of the catalysts of choice
for peptide bond formation because of its high reactivity and
its low degree of peptide epimerization, was by far not the
best coupling catalyst in our esterification reactions. It
leucine-based oligomers, differing in the stereochemistry of
the backbone repeat units and their connecting motifs. Due
to the replacement of amide bonds by ester-alt-urea moieties,
a significant change in hydrogen bonding pattern and hence
resulting structure formation was anticipated. Therefore, the
eminent structural differences of these compounds were
expected to largely affect their intermolecular interactions
in solution, leading to a significantly altered aggregation
behavior. Note that the present oligomers are of insufficient
(14) Moore, J. S.; Stupp, S. I. Macromolecules 1990, 23, 65.
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Hartwig et al.
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in their spectra upon changing the concentration.15 As a
consequence of the limited length of the oligomers all proton
signals are well separated and can be assigned by using
COSY and NOESY measurements, carried out at concen-
trations below the aggregation threshold (7.5 mmol/L).16
Once every relevant proton in the spectrum was correctly
attributed to the corresponding signal, aggregation could be
monitored by the downfield shift of the amide or urea
protons over a concentration range from 7.5 to 120 mmol/L,17
providing insight into both the overall aggregation tendency
as well as the specific interacting regions of the molecules.
Note that higher levels of hierarchical self-assembly, such
as fiber formation typically resulting in gelation, occur at
even higher concentrations and are not the subject of the
presented study.
The choice of the solvent for these studies is crucial for
several reasons. On the one hand, protons involved in the
aggregation process, which is primarily mediated by hydro-
gen bonding, are all exchanging protons and thus protic
solvents, such as water or alcohols, leading to fast proton
exchange could not be used. On the other hand, the interac-
tion of the compounds with the solvent should not disfavor
aggregation and hence, hydrogen bond competitors, such as
strongly solvating DMF or DMSO, had to be excluded.
Furthermore, the solvent should be capable of dissolving all
compounds even at higher concentrations, eliminating aceto-
nitrile as well as all apolar solvents, such as hexane or
toluene. Considering all these restrictions, CD2Cl2 was
the solvent of choice18 for the aggregation studies, although
excluding variable-temperature experiments well above
25 °C.
The concentration series of the peptides shows a markedly
different behavior for the all-(L)-tetrapeptide 11a as com-
pared to the (D)-alt-(L)-tetrapeptide 11b (Figure 4). Clearly,
11a experiences much more pronounced downfield shifts as
compared to 11b, suggesting much stronger association.
When looking at the individual shifts, 11b displays very
similar behavior for all four protons. In contrast, the inner
amide protons labeled 2 and 3 (as well as the carbamate
proton 1) of 11a exhibit larger chemical shift differences than
the outer amide proton 4, pointing to particular stabilization
of the aggregate by formation of hydrogen bonds in the core.
In the case of 11a, the terminal benzylic protons display a
characteristic splitting at high concentration, while in 11b
these benzylic protons are split even at low concentration
and one may therefore speculate about local conformational
restriction within 11a, for example, by loop formation.
Contrasting the studies of the tetrapeptides, the concen-
tration-dependent spectra of the partial ester-urea series, in
general, show smaller shift differences (Figure 5). Again,
more pronounced downfield shifts were observed for the
all-(L)-tetramer 10a as compared to the (D)-alt-(L)-analogue
10b. Similar to the peptide case, the four protons in 10a
exhibit rather different shifts of their signals in comparison
to 10b. Notably, one of the urea protons (3) in 10a experiences
FIGURE 4. 1H NMR spectra of (a) all-(L)-tetrapeptide 11a and (b)
(D)-alt-(L)-tetrapeptide 11b at varying concentrations. (c) Proton
chemical shift differences as a function of concentration for peptides
11a and 11b. All spectra were recorded in CD2Cl2 at 25 °C.
chain length to allow for intramolecular secondary structure
formation, in particular folding into helical structures.
NMR Aggregation Studies. To investigate the aggregation
of the various oligomers, NMR spectroscopy constitutes a
versatile tool, since the protons involved in hydrogen bond-
ing interactions are expected to display characteristic shifts
(16) Below this concentration (7.5 mmol/L), 1H NMR chemical shifts
remained constant, indicating the absence of intermolecular interaction, i.e.,
hydrogen bonds, and hence exclude aggregation.
(15) (a) Meraldy, J. P.; Hruby, V. J. J. Am. Chem. Soc. 1976, 98, 6408.
(b) Higashijima, T.; Tasumi, M.; Miyazawa, T.; Miyoshi, M. Eur. J.
Biochem. 1978, 89, 543. (c) Iqbal, M.; Balaram, P. Biochemistry 1981, 20,
7278. (d) Iqbal, M.; Balaram, P. Biopolymers 1982, 21, 1427. (e) Raj, P. A.;
Balaram, P. Biopolymers 1985, 24, 1131.
(17) Larger concentrations could hardly be realized as the solutions
became rather viscous.
(18) Due to its potential decomposition liberating HCl, CDCl3 was not
used as an alternative solvent as the aggregation process should be pH-
dependent.
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FIGURE 5. 1H NMR spectra of (a) all-(L)-tetramer 10a and (b) (D)-
alt-(L)-tetramer 10b at varying concentrations. (c) Proton chemical
shift differences as a function of concentration for peptides 10a and
10b. All spectra were recorded in CD2Cl2 at 25 °C.
FIGURE 6. 1H NMR spectra of (a) all-(L)-tetramer 7a and (b) (D)-
alt-(L)-tetramer 7b at varying concentrations. (c) Proton chemical
shift differences as a function of concentration for peptides 7a and
7b. All spectra were recorded in CD2Cl2 at 25 °C.
a much larger shift as compared to the other urea proton (2),
suggesting that the urea moiety engages in a nonsymmetric
hydrogen bond. The same is true for the urea protons in 10b;
however, the chemical shift differences are much smaller.
Last but not least, inspection of the concentration-depen-
dent NMR spectra of the exclusive ester-urea series reveals
that the shift differences are of the same magnitude for
both the all-(L)-tetramer 7a and the (D)-alt-(L)-tetramer 7b
(Figure 6). In both cases, the chemical shift differences are
comparable to those of 10a, suggesting a similar association
strength. When analyzing the contributions of the individual
protons, both urea moieties in 7b seem to be involved in
rather symmetrical hydrogen bonding interactions, as the
chemical shifts of protons 4 and 5 as well as 2 and 3 are nearly
identical. In contrast, one of the urea moieties in 7a shows
rather distinct chemical shift differences (protons 2 and 3),
while the other urea moiety is associated with equal chemical
shift differences (protons 4 and 5). Hence, the urea units in 7a
seem to engage in both symmetrical as well as nonsymme-
trical hydrogen bonding interactions.
The aggregation tendencies of the six investigated tetra-
mers can be summarized as follows: The native, natural
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Hartwig et al.
JOCArticle
compared to the (D)-alt-(L)-configured compounds. A poten-
tial explanation for the significantly reduced influence of the
configuration on the observed proton shift with increasing
amount of ester-alt-urea moieties involves commensurability
of hydrogen bonding donor and acceptor sites as well as
rotational stiffness of the backbones (Figure 7). The conforma-
tional space of the peptide backbone is governed by only two
angles φ and ψ (Ramachandran diagram), since the preferred
anti-conformation of the amide bond fixes this angle to
180°.20 This limited rotational freedom of the peptide back-
bone may inhibit the formation of a structure with reduced
steric interaction, which is still able to associate in a sheet-
type structure. This could explain the notable different
aggregation behavior for all-(L)- and (D)-alt-(L)-peptides.
The major difference between the peptide and the ester-alt-
urea structures is the enhanced rotational freedom of the
backbone. By incorporating the β-amino alcohol as well
as the ester units, four additional variable rotational angles
(R, β, γ, and δ) are introduced and significantly increase
backbone flexibility. This could explain the largely reduced
aggregation tendency in the partial and exclusive ester-urea
series due to a lower degree of preorganization of the back-
bone on the one hand, and the vanishing influence of
monomer configuration (stereochemistry) on association
on the other hand.
FIGURE 7. Comparison of peptide and ester-alt-urea backbones
with regard to conformational freedom and hydrogen bonding
pattern. Hydrogen bonding donor and acceptor sites are indicated
with green arrows and show the commensurability in the case of the
peptide only. In the peptide, rotational angles φ and ψ are displayed
in red, while in the case of ester-alt-ureas additional rotational
angles R, β, γ, and δ are displayed in blue. Fixed planes of the amide
and urea moieties are indicated with black boxes.
peptide 11a shows the most pronounced aggregation. Chan-
ging the stereochemistry from all-(L) to (D)-alt-(L) leads to a
significantly reduced aggregation of the peptide by a factor
of more than 3.19 Replacing two amide bonds of the natural
peptide by one ester-alt-urea fragment lowers the aggrega-
tion tendency of the naturally configured compound 10a by a
factor of more than 2 (in comparison to 11a). Within the
partial ester-urea series, a change of stereochemistry from
all-(L) to (D)-alt-(L) (10a f 10b) again leads to a notable, yet
less pronounced drop of aggregation by a factor of roughly
1.5. Finally, substituting all amide bonds with alternating
ester and urea moieties leads to a further, but weak decrease
of aggregation (by a factor of 1.3, comparing 7a with 10a).
Importantly, within the complete ester-alt-urea series, stereo-
chemical variation has negligible influence on aggregation.
Therefore, replacing dipeptide units by an ester-alt-urea
isostere not only reduces the intrinsic aggregation tendency,
but also decreases the influence of stereochemistry on the
aggregation behavior of the corresponding oligomer.
As no detailed structural data are available at the moment,
a discussion of these results is rather speculative. However,
assuming a β-sheet-type aggregate20 structure for all com-
pounds, the different proton shifts have to result from
intrinsic strengths of the different hydrogen bonds (amide
vs urea) and from different steric interactions of the side
chain residues. The all-(L)-configured compounds should
experience much weaker steric repulsion in this assumed
aggregate structure, resulting in stronger association as
Conclusion
The ester-urea motif was incorporated into the peptide
backbone to replace two adjacent amide residues, giving rise
to two new peptide-mimicking scaffolds with either 50% or
100% ester-urea content. Furthermore, the stereochemistry
was varied between all-(L) and (D)-alt-(L). The synthesis of
different short oligomers, i.e., tetramers 7a/7b and 10a/10b,
was readily accomplished by using ester as well as amide
couplings to key urea building blocks 3a/3b. After full
characterization, the thus prepared peptide mimics were
investigated with regard to their aggregation behavior in
organic solution by means of concentration-dependent
NMR spectroscopy. It turns out that the naturally occurring
peptide scaffold shows the largest association tendency and
also the largest discrimination is observed between the all-(L)
and (D)-alt-(L) stereoisomers. Increasing incorporation of
the ester-urea motif decreases the aggregation tendency and
also the stereochemical differentiation becomes less pro-
nounced and finally vanishes at the complete ester-urea
series.
Our findings point to some important structural details in
peptides and re-emphasize key requirements for (their) effi-
cient association processes. Although, the urea moiety itself
is rigid and engages in rather strong hydrogen bonding
interactions, introduction of the ester reduces the number
of interacting sites and furthermore increases conforma-
tional flexibility due to employed β-amino alcohol unit and
the ester linkage. Both of these effects significantly lower the
overall association tendency and ability of stereochemical
differentiation of the backbone configuration. Clearly, the
correct orientation of amide bonds is most suited to mediate
association. On the one hand, amides offer the advantage to
display an equal number of hydrogen bonding donating and
accepting sites to maximize the number of interacting units
on both faces of the backbone following the principles of
(19) This factor is based on the average chemical shift differences of all
participating protons (numbered in Figures 5-7). Due to the limited con-
centration range and the significant deviations of the chemical shift differ-
ences of the individual protons, no association constants were calculated as
these individual or average values would have rather limited meaning. True
association constants necessitate, for example, in depth isothermal titration
calorimetry measurements.
(20) (a) Sewald, N.; Jakubke, H.-D. Peptides: Chemistry and Biology;
Wiley-VCH: Weinheim, Germany, 2003. (b) Quinkert, G.; Egert, E.; Griesinger,
C. Aspects of Organic Chemistry: Structure; VHCA, Basel, Switzerland, 1996.
778 J. Org. Chem. Vol. 75, No. 3, 2010

Hartwig et al.
JOCArticle
commensurability and multivalency.21 On the other hand,
they are conformationally restricted and hence their preor-
ganization leads to a favorable (or at least not negative)
entropic contribution to association.22 Interestingly, association
of the peptide is much more dependent on the backbone
configuration (as compared to the ester-ureas), demonstrating
the large effect of stereochemistry on backbone conforma-
tion and the ability of using side chain interactions to tune
peptide association. The obtained structure-aggregation
relationships illustrate the emergence and particular strength
of β-sheet-type aggregation in peptides and proteins23 and
point to potential ways to weaken their association processes
by using β-sheet breaking peptide-mimics24 and to design
improved small molecule gelators.8
Future work will be focused on detailed investigations
elucidating the structure of the formed aggregates and their
superstructures, i.e., fibers/gels, as well as determination of
the association constants by isothermal titration calorime-
try. Further synthetic work will be targeting the design of
water-soluble peptide mimics with incorporated photo-
switchable units to externally control self-assembly.25
(PE: EA = 2: 8). HPLC (2 ꢀ 150 mm Luna Phenyl-Hexyl 3 μm,
acetonitrile (AN):water, grad: 5 f 95 vol % AN): tR = 18.57
min (>99.0% peak area). 1H NMR (300 MHz, CDCl3, 20 °C):
δ 7.36-7.31 (m, 5 H, C11-13H), 5.99-5.94 (m, 1 H, N6H),
5.59-5.54 (m, 1 H, N4H), 5.19-5.07 (m, 2 H, C9H2), 4.48-4.42
(m, 1 H, C7H), 4.00 (br s, 1 H, O1H), 3.76-3.71 (m, 1 H, C3H),
3.58 (dd, 2J(H,H) = 11.0 Hz, 3J(H,H) = 3.4 Hz, 1 H, 1 C2H2),
3.46 (dd, 2J(H,H) = 11.0 Hz, 3J(H,H) = 6.5 Hz, 1 H, 1 C2H2),
1.72-1.46 (m, 4 H, C14H2, C17H2), 1.44-1.23 (m, 2 H, C15H,
C18H), 0.93-0.87 (m, 12 H, 2 C16H3, 2 C19H3). 13C NMR
(CDCl3): δ 174.6, 159.2, 135.5, 128.5, 128.3, 128.0, 66.9, 66.8,
52.0, 51.1, 41.5, 40.5, 24.81, 24.78, 23.1, 22.8, 22.2, 22.0.
HR-ESI-MS: m/z 365.2435 (calcd 365.2443 for C20H32N2O4 þ
1 Hþ).
(L)-Leucinol-urea-(D)-leucine-Bn (3b). (D)-Leu-Bn 1a (13.08 g,
39.00 mmol) and NEt3 (7.59 mL, 54.60 mmol) were dissolved in
CH2Cl2 (100 mL) and added to a solution of CDI (12.65 g,
78.00 mmol) in CH2Cl2 (400 mL) at 0 °C within 1 h. After
addition, the mixture was stirred for 1 h at 0 °C, then for 2 h at
room temperature. The reaction mixture was extracted with
water (2 ꢀ 100 mL). The organic layer was dried over MgSO4,
filtered, and evaporated in vacuo to give the intermediate
imidazole-urea. (L)-Leucinol 2 (4.80 g, 40.95 mmol), dissolved
in CH2Cl2 (400 mL), was cooled to 0 °C and the intermediate
imidazole-urea, dissolved in CH2Cl2 (100 mL), was added with-
in 1 h. The mixture was allowed to warm to room temperature
and stirred for 16 h. Water was added to the reaction mixture
and the biphasic system was stirred for 30 min. After phase
separation, the organic layer was evaporated in vacuo and
CH2Cl2 was replaced by EA. The organic layer was washed
with water (2 ꢀ 100 mL), 1 M aqueous citric acid solution (2 ꢀ
50 mL), water (1 ꢀ 50 mL), saturated aqueous NaHCO3
solution (1 ꢀ 50 mL), water (1 ꢀ 50 mL), and brine (1 ꢀ
50 mL). The organic layer was dried over MgSO4, filtered,
and evaporated in vacuo to give the crude product, which was
purified via recrystallization from PE:CH2Cl2 to give 12.51 g
(88%) of pure 3b as colorless crystals. Rf 0.50 (PE: EA = 2: 8).
HPLC (2 ꢀ 150 mm Luna Phenyl-Hexyl 3 μm, acetonitrile:
water, grad: 5 f 95 vol % AN): tR = 18.82 min (>99.0% peak
area). 1H NMR (300 MHz, CDCl3, 20 °C): δ 7.34-7.30 (m, 5 H,
Experimental Section
The synthesis and characterization data of key compounds
are provided below. For further details on general methods,
experimental procedures, and characterization data, including
copies of spectral data, consult the Supporting Information.
(L)-Leucinol-urea-(L)-leucine-Bn (3a). (L)-Leu-Bn 1a (1.01 g,
3.00 mmol) and NEt3 (0.42 mL, 3.00 mmol) were dissolved in
CH2Cl2 (20 mL) and added to a solution of carbonyldiimidazole
(CDI; 0.97 g, 6.00 mmol) in CH2Cl2 (50 mL) at 0 °C within 1 h.
After being stirred at 0 °C for 1 h, the mixture was allowed to
warm to room temperature and stirred for 1 h. The reaction
mixture was extracted with water (2 ꢀ 100 mL) and brine (1 ꢀ
100 mL). The organic layer was dried over MgSO4, filtered, and
evaporated in vacuo to give the intermediate imidazole-urea.
L-Leucinol 2 (0.35 g, 3.00 mmol), dissolved in CH2Cl2 (100 mL),
was cooled to 0 °C and the intermediate imidazole-urea, dis-
solved in CH2Cl2 (20 mL), was added within 20 min. The
mixture was allowed to warm to room temperature and stirred
for 16 h. The reaction mixture was washed with water (1 ꢀ 50
mL), 1 M aqueous citric acid solution (1 ꢀ 50 mL), water (1 ꢀ 50
mL), saturated aqueous NaHCO3 solution (1 ꢀ 50 mL), water
(1 ꢀ 50 mL), and brine (1 ꢀ 50 mL). The organic layer was dried
over MgSO4, filtered, and evaporated in vacuo to give 1.16 g of
the crude product, which was purified via silica column chro-
matography (eluent: PE:EA = 1:1). The second column gave
900 mg (82%) of pure product as colorless crystals. Rf 0.50
3
C11-13H), 6.01 (d, J(H,H) = 6.7 Hz, 1 H, N6H), 5.66-5.60
(m, 1 H, N4H), 5.20-5.05 (m, 2 H, C9H2), 4.56-4.44 (m, 1 H,
C7H), 4.17 (br s, 1 H, O1H), 3.85-3.70 (m, 1 H, C3H), 3.60 (dd,
2J(H,H) = 11.0 Hz, 3J(H,H) = 3.2 Hz, 1 H, 1 C2H2), 3.41 (dd,
2J(H,H) = 11.0 Hz, 3J(H,H) = 5.8 Hz, 1 H, 1 C2H2), 1.79-1.46
(m, 4 H, C14H2, C17H2), 1.44-1.17 (m, 2 H, C15H, C18H),
0.93-0.87 (m, 12 H, C16H3, C19H3). 13C NMR (CDCl3):
δ 175.2, 158.9, 135.4, 128.5, 128.2, 128.0, 66.9, 66.1, 51.8, 50.5,
41.5, 40.8, 24.8, 23.0, 22.8, 22.3, 21.9. HR-ESI-MS: m/z
365.2456 (calcd 365.2440 for C20H33N2O4 þ 1 Hþ).
TBDPS-(L)-leucinol-urea-(L)-leucine-Bn (4a). 3a (5.45 g,
14.95 mmol), imidazole (2.04 g, 29.90 mmol), and TBDPS-Cl
(0.17 g, 1.12 mmol) were dissolved in DMF (50 mL) and the
mixture was stirred for 3 h. The solution was poured on ice, and
the aqueous layer was extracted with Et2O, dried over MgSO4,
and evaporated in vacuo. The crude product was purified via
silica column chromatography (eluent: PE:EA = 10:1) to give
7.38 g (yield: 81%) of the desired product. An NMR sample in
CDCl3 showed decomposition but the pure product could be
stored in a freezer without decomposition. Rf 0.70 (PE:EA =
10:1). UPLC (2.1 ꢀ 100 mm BEH Phenyl 1.7 μm, acetonitrile:
water, grad: 40 f 95 vol % AN): tR = 4.60 min (86.1% peak
area). 1H NMR (300 MHz, CD2Cl2, 20 °C): δ 7.72-7.56 (m, 4 H,
C19H), 7.38-7.25 (m, 11 H, C12-14H, C20-21H), 5.10 (s, 2 H,
C10H2), 4.67 (d, 3J(H,H) = 8.7 Hz, 1 H, N7H), 4.46 (d, 3J(H,H) =
9.1 Hz, 1 H, N5H), 4.43-4.34 (m, 1 H, C5H), 3.84-3.71 (m, 1 H,
C4H), 3.65-3.51 (m, 1 H, C3H2), 1.67-1.25 (m, 6 H, 2 C15H2, 2
(21) (a) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int.
Ed. 1998, 37, 2754. (b) Lundquist, J. L.; Toone, E. J. Chem. Rev. 2002, 102,
555.
(22) (a) Page, M. L.; Jencks, W. P. Proc. Natl. Acad. Sci. 1971, 68, 1678.
For a review, see: (b) Jencks, W. P. Adv. Enzymol. 1975, 43, 219. A theoretical
treatment is given in: (c) Mammen, M.; Shaknovich, E. I.; Whitesides, G. M.
J. Org. Chem. 1998, 63, 3168.
(23) (a) Rauk, A. Chem. Soc. Rev. 2009, 38, 2698. (b) Hamley, I. W.
Angew. Chem., Int. Ed. 2007, 46, 8128. (c) Chiti, F.; Dobson, C. M. Annu.
Rev. Biochem. 2006, 75, 333. (d) Misbehaving Proteins: Protein (Mis)Folding,
Aggregation, and Stability, Tsai, A.; Murphy, R., Ed.; Springer: Berlin, Germany,
2006.
(24) For recent examples, see: (a) Coelho, M.; Rocha, S. Biochem.
Biophys. Res. Commun. 2009, 380, 397. (b) Kim, Y. S.; Lim, D.; Kim,
J. Y.; Kang, S. J.; Kim, Y.-H.; Im, H. Biochem. Biophys. Res. Commun.
2009, 387, 682. For overviews, see: (c) Adessi, C.; Soto, C. Drug Dev. Res.
2002, 56, 184. (d) Mason, J. M.; Kokkoni, N.; Stott, K.; Doig, A. J. Curr.
Opin. Struct. Biol. 2003, 13, 526.
(25) For a highlight, see: Hecht, S. Small 2005, 1, 26 and references cited
therein.
C16H), 1.00 (s, 9 H, 3 C1H3), 0.91-0.74 (m, 12 H, 4 C17H3). 13
J. Org. Chem. Vol. 75, No. 3, 2010 779
C

Hartwig et al.
JOCArticle
NMR (CD2Cl2): δ 174.0, 156.9, 135.9, 135.6, 133.6, 133.5, 129.7,
129.7, 128.5, 128.2, 128.0, 127.7, 66.6, 66.5, 51.8, 49.8, 41.8, 41.2,
26.7, 24.8, 24.7, 22.8, 22.6, 21.6, 20.8, 19.2. HR-ESI-MS: m/z
603.3449 (calcd 603.3618 for C36H51N2O4Si1 þ 1 Hþ).
vacuo. The residue was purified via silica column chromatog-
raphy (eluent: PE:EA = 4:2) to give 1.95 g (yield: 55%) of the
desired product. Rf 0.50 (PE:EA = 2:1). UPLC (2.1 ꢀ 100 mm
BEH Phenyl 1.7 μm, acetonitrile:water, grad: 40 f 95 vol %
1
TBDPS-(L)-leucinol-urea-(D)-leucine-Bn (4b). 3b (5.45 g,
14.95 mmol), imidazole (2.04 g, 29.90 mmol), and TBDPS-Cl
(0.17 g, 1.12 mmol) were dissolved in DMF (50 mL) and the
mixture was stirred for 3 h. The solution was poured on ice, and
the aqueous layer wasextracted with Et2O, dried over MgSO4,
and evaporated in vacuo. The crude product was purified via
silica column chromatography (eluent: PE:EA=10:1) to give
7.63 g (yield: 85%) of the desired product. An NMR sample in
CDCl3 showed decomposition but the pure product could be
stored in a freezer without decomposition. Rf 0.70 (PE:EA =
10:1). HPLC (2 ꢀ 150 mm Luna Phenyl-Hexyl 3 μm, acetoni-
trile:water, grad: 40 f 95 vol % AN): tR = 20.02 min (>99.0%
peak area). 1H NMR (300 MHz, CD3CN, 20 °C) δ 7.70-7.67
(m, 4 H, 4 C19H), 7.45-7.34 (m, 11 H, C12-14H, 2 C20-21H), 5.31
AN): tR = 5.61 min (84.0% peak area). H NMR (300 MHz,
CDCl3, 20 °C): δ 7.67-7.62 (m, 4 H, C26H), 7.43-7.28 (m, 10 H,
C19-21H, C27-28H), 5.42-5.26 (m, 2 H, 2 NH), 5.15-4.90 (m, 3 H,
C17H2, NH), 4.73-4.60 (m, 1 H, NH), 4.57-4.42 (m, 2 H, C8H,
C15H), 4.35-4.24 (m, 1 H, C11H), 4.07-3.93 (m, 1 H, C4H),
3.95-3.57 (m, 4 H, C3H2, C10H2), 1.73-1.24 (m, 12 H, 4 C22H2,
4 C23H), 1.07 (s, 9 H, 3 C1H3), 0.98-0.82 (m, 24 H, 8 C24H3). 13
C
NMR (CDCl3): δ 174.2, 157.8, 157.3, 135.6, 133.4, 133.3,
129.84, 129.80, 128.5, 128.2, 128.1, 127.8, 66.9, 66.7, 50.6,
52.2, 51.6, 47.3, 42.3, 41.4, 41.2, 41.0, 26.9, 24.76, 24.72, 22.9,
22.8, 22.7, 22.5, 22.4, 22.1, 19.3, 14.9. HR-ESI-MS: m/z
859.5394 (calcd 859.5400 for C49H75N4O7Si1 þ 1 Hþ).
TBDPS-[(L)-leucinol-urea-(D)-leucine]2-Bn (6b). 3b (1.50 g,
4.12 mmol), 5b (2.32 g, 4.53 mmol), and DMAP (0.50 g,
4.12 mmol) were dissolved in CH2Cl2 (100 mL) and the mixture
was cooled to 0 °C. EDC (1.74 g, 9.05 mmol), dissolved in
CH2Cl2 (5 mL), was added slowly. The reaction mixture was
allowed to warm to room temperature, stirred for 5 h, and
evaporated in vacuo. The residue was purified via silica column
chromatography (eluent: PE:EA = 4:2) to give 1.88 g (yield:
53%) of the desired product. Rf 0.50 (PE:EA = 2:1). HPLC (2 ꢀ
150 mm Luna Phenyl-Hexyl 3 μm, acetonitrile:water, grad: 70 f
3
(d, J(H,H) = 8.3 Hz, 1 H, N7H), 5.12 (s, 2 H, C10H2), 5.05
3
(d, J(H,H) = 8.3 Hz, 1 H, N5H), 4.39-4.31 (m, 1 H, C5H),
3.86-3.82 (m, 1 H, C4H), 3.61-3.58 (m, 1 H, C3H2), 1.66-1.37
(m, 6 H, 2 C15H2, 2 C16H), 1.06 (s, 9 H, 3 C1H3), 0.98-0.83 (m,
12 H, 4 C17H3). 13C NMR (CD3CN): δ 174.6, 158.2, 137.3,
136.4, 134.5, 134.5, 130.8, 130.7, 129.4, 129.0, 128.9, 128.8, 67.5,
68.0, 52.6, 50.3, 42.1, 41.8, 27.2, 25.6, 25.6, 23.5, 23.1, 22.6, 22.1,
19.8. HR-ESI-MS: m/z 603.3604 (calcd 603.3613 for
C36H51N2O4Si1 þ 1 Hþ).
1
95 vol % AN): tR = 15.75 min (95.8% peak area). H NMR
(300 MHz, CD3CN, 20 °C): δ 7.70-7.64 (m, 4 H, C26H),
TBDPS-(L)-leucinol-urea-(L)-leucine (5a). To a solution of 4a
(1.00 g, 1.60 mmol) and 50 mL of ethanol was added Pd/C (10 wt %,
120 mg) and the solution was stirred under hydrogen atmo-
sphere at room temperature for 12 h. The reaction mixture was
filtered and evaporated in vacuo to give the desired product in
55% yield (0.47 g). Rf 0.50 (CH2Cl2:MeOH =100: 5). UPLC
(2.1 ꢀ 100 mm BEH Phenyl 1.7 μm, acetonitrile:water, grad:
40 f 95 vol % AN): tR = 4.86 min (68.0% peak area). 1H NMR
(300 MHz, CD2Cl2, 20 °C): δ 7.71-7.65 (m, 4 H, C15H),
7.50-7.37 (m, 6 H, C16-17H), 5.34-5.24 (m, 1 H, N7H),
5.20-5.07 (m, 1 H, N5H), 4.30-4.20 (m, 1 H, C5H),
3.90-3.75 (m, 1 H, C4H), 3.71-3.59 (m, 1 H, C3H2),
1.81-1.31 (m, 6 H, 2 C11H2, 2 C12H), 1.09 (s, 9 H, 3 C1H3),
1.04-0.88 (m, 12 H, 4 C13H3). 13C NMR (CD2Cl2): δ 175.7,
159.0, 135.6, 133.4, 133.2, 129.8, 128.5, 128.0, 127.7, 66.7, 60.3,
40.8, 40.4, 26.7, 24.72, 24.68, 22.77, 22.76, 22.0, 21.5, 20.0. HR-
ESI-MS: m/z 513.3134 (calcd 513.3143 for C29H45N2O4Si1 þ
1 Hþ).
3
7.45-7.37 (m, 10 H, C19-21H, C27-28H), 5.43 (d, J(H,H) =
7.2 Hz, 1 H, NH), 5.32 (d, 3J(H,H) = 7.6 Hz, 1 H, NH),
5.23-5.04 (m, 4 H, C17H2, 2 NH), 4.35-4.12 (m, 3 H, C8H,
C11H, C15H), 3.95-3.76 (m, 3 H, C4H, C10H2), 3.64-3.51 (m, 1 H,
C3H2), 1.72-1.16 (m, 12 H, 4 C22H2, 4 C23H), 1.04 (s, 9 H,
3 C1H3), 0.94-0.74 (m, 24 H, 8 C24H3). 13C NMR (CD3CN): δ
173.7, 173.3, 157.8, 157.6, 136.3, 135.5, 133.5, 133.4, 129.82,
129.79, 128.5, 128.1, 127.9, 127.8, 117.3, 66.8, 66.5, 66.2, 52.1,
51.7, 49.4, 47.0, 41.05, 40.98, 40.9, 40.7, 26.3, 24.6, 24.6, 24.5,
22.6, 22.4, 22.2, 21.6, 21.4, 21.15, 21.09, 18.9. HR-ESI-MS: m/z
859.5402 (calcd 859.5400 for C49H75N4O7Si1 þ 1 Hþ).
[(L)-Leucinol-urea-(L)-leucine]2-Bn (7a). 6a(0.22 g, 0.26 mmol)
was dissolved in acetonitrile (10 mL) and HF (0.10 mL) was
added. The reaction mixture was stirred for 2 h. K2CO3 was
added, and the solution was filtered and evaporated in vacuo.
The residue was purified via silica column chromatography
(eluent: CH2Cl2:MeOH = 10:1) to give 0.15 g (yield: 94%) of
the desired product. Rf 0.50 (CH2Cl2:MeOH = 10:1). UPLC
(2.1 ꢀ 100 mm BEH Phenyl 1.7 μm, acetonitrile:water, grad:
40 f 95 vol % AN): 3.45 min (>99.9% peak area). 1H NMR
(400 MHz, CD2Cl2, 20 °C): δ 7.40-7.29 (m, 5 H, C18-20H), 5.46
(br s, 1 H, N6H), 5.35 (d, 3J(H,H) = 8.2 Hz, 1 H, N13H), 5.27
TBDPS-(L)-leucinol-urea-(D)-leucine (5b). To a solution of 4b
(1.00 g, 1.60 mmol) in 50 mL of ethanol was added Pd/C (10 wt
%, 120 mg) and the solution was stirred under hydrogen atmo-
sphere at room temperature for 12 h. The reaction mixture was
filtered and evaporated in vacuo to give the desired product in
58% yield (0.49 g). Rf 0.50 (CH2Cl2:MeOH = 100:5). HPLC
(2 ꢀ 150 mm Luna Phenyl-Hexyl 3 μm, acetonitrile:water, grad:
3
(d, J(H,H) = 9.0 Hz, 1 H, N11H), 5.12 (s, 2 H, C16H2), 4.78
(d, 3J(H,H) = 7.5 Hz, 1 H, N4H), 4.48 (dd, 2J(H,H) = 10.8 Hz,
1
3
3J(H,H) = 3.4 Hz, 1 H, 1 C9H2), 4.41 (dt, J(H,H) = 8.5 Hz,
40 f 95 vol % AN): tR = 17.18 min (>99.9% peak area). H
NMR (300 MHz, CD3CN, 20 °C): δ 7.70-7.66 (m, 4 H, C15H),
7.45-7.42 (m, 6 H, C16-17H), 5.42 (d, 3J(H,H) = 7.1 Hz, 1 H,
N7H), 5.29 (d, 3J(H,H) = 8.3 Hz, 1 H, N5H), 4.20-4.16 (m, 1 H,
C5H), 3.80-3.77 (m, 1 H, C4H), 3.63-3.60 (m, 1 H, C3H2),
1.81-1.31 (m, 6 H, 2 C11H2, 2 C12H), 1.06 (s, 9 H, 3 C1H3),
0.91-0.86 (m, 12 H, 4 C13H3). 13C NMR (CD3CN): δ 174.5,
158.7, 135.5, 129.8, 129.8, 127.8, 66.4, 52.1, 49.7, 40.6, 40.3, 26.3,
24.6, 24.6, 22.5, 22.2, 21.6, 21.0, 18.9. HR-ESI-MS: m/z
513.3134 (calcd 513.3143 for C29H45N2O4Si1 þ 1 Hþ).
3J(H,H) = 6.0 Hz, 1 H, C14H), 4.16 (dt, 3J(H,H) = 8.5 Hz, 3J(H,
H) = 6.0 Hz, 1 H, C7H), 4.02-3.92 (m, 1 H, C10H), 3.81 (dd,
2J(H,H) = 10.8 Hz, 3J(H,H) = 4.0 Hz, 1 H, 1 C9H2), 3.70-3.61
(m, 1 H, C3H), 3.60-3.54 (m, 1 H, 1 C2H2), 3.47-3.34 (m, 2 H, 1
C2H2, O1H), 1.74-1.19 (m, 12 H, 4 C21H2, 4 C22H), 0.97-0.87
(m, 24 H, 8 C23H3). 13C NMR (CD2Cl2): δ 174.4, 174.1, 159.3,
157.1, 135.8, 128.5, 128.2, 128.0, 67.2, 66.7, 52.4, 52.8, 47.1, 41.9,
41.5, 41.1, 40.8, 40.3, 24.9, 24.8, 24.1, 22.9, 22.6, 22.54, 22.52,
22.1, 22.0, 21.9, 21.8. HR-ESI-MS: m/z 621.4229 (calcd
621.4222 for C33H56N4O7 þ 1 Hþ).
TBDPS-[(L)-leucinol-urea-(L)-leucine]2-Bn (6a). 3a (1.50 g,
4.12 mmol), 5a (2.32 g, 4.53 mmol), and DMAP (0.50 g, 4.12
mmol) were dissolved in CH2Cl2 (100 mL) and the mixture was
cooled to 0 °C. EDC (1.74 g, 9.05 mmol), dissolved in CH2Cl2
(5 mL), was added slowly. The reaction mixture was allowed to
warm to room temperature, stirred for 5 h, and evaporated in
[(L)-Leucinol-urea-(D)-leucine]2-Bn (7b). 6b (0.22 g, 0.26 mmol)
was dissolved in acetonitrile (10 mL) and HF (0.10 mL) was
added. The reaction mixture was stirred for 2 h. K2CO3 was
added, and the solution was filtered and evaporated in vacuo.
The residue was purified via silica column chromatography
780 J. Org. Chem. Vol. 75, No. 3, 2010

Hartwig et al.
JOCArticle
(eluent: CH2Cl2:MeOH = 10:1) to give 0.16 g (yield: 98%) of the
desired product. Rf 0.50 (CH2Cl2:MeOH = 10:1). HPLC (2 ꢀ
150 mm Luna Phenyl-Hexyl 3 μm, acetonitrile:water, grad: 40 f
95 vol% AN): tR = 14.80 min (>99.9% peak area). 1H NMR
(400 MHz, CD2Cl2, 20 °C): δ 7.40-7.31 (m, 5 H, C18-20H), 5.16
(d, 3J(H,H) = 7.8 Hz, 1 H, N13H), 5.11 (d, 3J(H,H) = 8.2 Hz, 1
H, N6H), 5.15 (dd, 2J(H,H) = 51.3 Hz, 3J(H,H) = 12.4 Hz, 2 H,
C16H2), 4.89 (d, 3J(H,H) = 8.0 Hz, 1 H, N4H), 4.84 (d, 3J(H,H) =
9.4Hz, 1H, N11H), 4.39-4.31 (m, 2H, C7H, C14H), 4.25-4.20(m,
1 H, O1H), 4.13-4.02 (m, 2 H, 1 C9H2, C10H), 3.89-3.81
(m, 2 H, C3H, 1 C9H2), 3.67-3.61 (m, 1 H, 1 C2H2), 3.39-3.32
(m, 1H, 1C2H2), 1.81-1.18(m, 12H, 4C21H2, 4C22H), 0.97-0.87
(m, 24 H, 8 C23H3). 13C NMR (CD2Cl2): δ 174.7, 173.4, 159.6,
158.1, 135.6, 128.5, 128.0, 67.9, 66.9, 66.6, 53.8, 53.4, 53.1, 50.7,
47.2, 40.84, 40.79, 40.6, 40.5, 24.8, 24.7, 22.9, 22.8, 22.7, 22.6, 22.1,
21.8. HR-ESI-MS: m/z 621.4230 (calcd 621.4222 for C33H56N4O7
þ 1 Hþ).
was added Pd/C (10 wt %, 170 mg) and the solution was stirred
under 6 bar hydrogen-pressure atmosphere at room tempera-
ture for 1 h. The reaction mixture was filtered and evaporated in
vacuo to give 1.45 g of the desired product (99% yield). Rf 0.40
(Et2O). HPLC (2 ꢀ 150 mm Luna Phenyl-Hexyl 3 μm, acetoni-
trile:water, grad: 5 f 95 vol % AN): tR = 19.84 min (>99.0%
1
peak area). H NMR (300 MHz, CD3OD, 20 °C): δ 4.29 (dd,
J(H,H) = 5.2 Hz, J(H,H) = 9.3 Hz, 1 H, C5H or C12H), 4.17
(t, 3J(H,H) = 7.5 Hz, 1 H, C5H or C12H or 1 C7H2), 4.11-3.96
(m, 3 H, 1 C7H2, C8H, 1 C7H2 or C5H or C12H), 1.83-1.25 (m,
18 H, 3 C1H3, 3 C15H2, 3 C26H), 0.97-0.91 (m, 18 H, 6 C17H3).
13C NMR (CD3OD): δ 177.2, 174.8, 160.1, 158.1, 80.5, 68.4,
53.5, 52.7, 48.1, 42.6, 42.1, 41.6, 28.8, 26.0, 25.8, 23.6, 23.4, 23.3,
22.5, 22.2, 21.9. HR-ESI-MS: m/z 488.3347 (calcd 488.3336 for
C24H45N3O7 þ 1 Hþ).
Boc-(D)-leucine-ester-(L)-leucinol-urea-(D)-leucine (9b). To a
solution of 8b (1.73 g, 3.00 mmol) in 50 mL of ethyl acetate was
added Pd/C (10 wt %, 170 mg) and the solution was stirred under
6 bar hydrogen-pressure atmosphere at room temperature for 3 h.
The reaction mixture was filtered and evaporated in vacuo to give
1.08 g of the desired product (74% yield). Rf 0.30 (Et2O). HPLC
(2 ꢀ 150 mm Luna Phenyl-Hexyl 3 μm, acetonitrile:water, grad:
5 f 95 vol % AN): tR = 20.15 min (>99.0% peak area). 1H NMR
(300MHz, CD3OD, 20 °C): δ4.32 (dd, J(H,H) =5.1Hz, J(H,H) =
9.3 Hz, 1 H, C5H or C12H), 4.16 (t, 3J(H,H) = 7.5 Hz, 1 H, C5H
or C12H or 1 C7H2), 4.06-4.01 (m, 3 H, 1 C7H2, C8H, 1 C7H2 or
C5H or C12H), 1.82-1.25 (m, 18 H, 3 C1H3, 3 C15H2, 3 C26H),
0.97-0.91 (m, 18 H, 6 C17H3). 13C NMR (CD3OD): δ 177.1,
174.7, 159.9, 158.1, 80.4, 68.4, 53.5, 52.6, 48.1, 42.8, 41.9, 41.3,
28.7, 26.0, 25.9, 25.8, 23.6, 23.4, 23.3, 22.4, 22.2, 21.8. HR-ESI-
MS: m/z 488.3347 (calcd 488.3336 for C24H45N3O7 þ 1 Hþ).
Boc-(L)-leucine-ester-(L)-leucinol-urea-(L)-leucine-amide-(L)-leu-
cine-Bn (10a). 9a (1.27 g, 2.61 mmol), 1a (0.60 g, 2.70 mmol), and
DMAP (0.32 g, 2.61 mmol) were dissolved in CH2Cl2 (30 mL)
and the mixture was cooled to 0 °C. To the cold solution was
added EDC (1.00 g, 5.22 mmol). The solution was allowed to
warm to room temperature and stirred for 24 h. The reaction
mixture was quenched by the addition of silica gel. After the
mixture was stirred for 4 h, the solvent was evaporated in vacuo.
The residue was purified via silica column chromatography
(eluent: PE:EA = 5:1 to 3:1) to give 1.52 g (84%) of pure 10a.
Rf 0.34 (PE:EA = 1:1). HPLC (2 ꢀ 150 mm Luna Phenyl-Hexyl
3 μm, acetonitrile:water, grad: 5 f 95 vol % AN): tR = 19.84 min
(97.0% peak area). 1H NMR (400 MHz, CD2Cl2, 20 °C): δ
7.40-7.30 (m, 5 H, C19-21H), 6.74 (d, 3J(H,H) = 7.6 Hz, 1 H,
N14H), 5.13 (dd, J(H,H) = 12.8 Hz, J(H,H) = 16.6 Hz, 2 H,
Boc-(L)-leucine-ester-(L)-leucinol-urea-(L)-leucine-Bn (8a). 3a
(1.09 g, 3.00 mmol), Boc-(L)-Leu-OH (0.90 g, 3.60 mmol), and
DMAP (0.37 g, 3.00 mmol) were dissolved in CH2Cl2 (250 mL)
and the mixture was cooled to 0 °C. To the cold solution was
added EDC (1.73 g, 1.80 mmol). The solution was allowed to
warm to room temperature and stirred for 2 h. The reaction
mixture was quenched by the addition of silica gel. After the
mixture was stirred for 2 h, the solvent was evaporated in vacuo
on a column and purified via silica column chromatography
(eluent: Et2O). The crude product (1.74 g) was purified via silica
column chromatography (eluent: PE:EA) to remove more un-
polar substances to give 1.72 g of pure 7a. Rf 0.28 (PE:EA 4:1).
HPLC (2 ꢀ 150 mm Luna Phenyl-Hexyl 3 μm, acetonitrile:
water, grad: 5 f 95 vol % AN): tR = 23.07 min (>99.0% peak
area). 1H NMR (300 MHz, CD3OD, 20 °C): δ 7.36-7.30 (m, 5 H,
C16-18H), 5.14 (dd, J(H,H) = 12.4 Hz, J(H,H) = 13.5 Hz, 2 H,
C14H2), 4.35 (dd, J(H,H) = 5.8 Hz, J(H,H) = 9.1 Hz, 1 H, C5H
3
or C12H), 4.17 (t, J(H,H) = 7.5 Hz, 1 H, C5H or C12H or 1
C7H2), 4.06-3.97 (m, 3 H, 1 C7H2, C8H, 1 C7H2 or C5H or
C12H), 1.78-1.24 (m, 18 H, 3 C1H3, 3 C19H2, 3 C20H),
0.97-0.89 (m, 18 H, 6 C21H3). 13C NMR (CD3OD): δ 175.1,
174.8, 159.9, 158.1, 135.2, 129.5, 129.20, 129.17, 80.5, 68.3, 67.6,
53.4, 53.0, 48.1, 42.2, 42.0, 41.6, 28.7, 25.9, 25.9, 25.7, 23.6, 23.3,
23.2, 22.4, 22.2, 21.9. HR-ESI-MS: m/z 578.3797 (calcd
578.3805 for C31H51N3O7 þ 1 Hþ).
Boc-(D)-leucine-ester-(L)-leucinol-urea-(D)-leucine-Bn (8b). 3b
(1.09 g, 3.00 mmol), Boc-(D)-Leu-OH (0.90 g, 3.60 mmol), and
DMAP (0.37 g, 3.00 mmol) were dissolved in CH2Cl2 (250 mL)
and cooled to 0 °C. To the cold solution was added EDC (1.73 g,
1.80 mmol). The solution was allowed to warm to room
temperature and stirred for 5 h. The reaction mixture was
quenched by the addition of silica gel. After the mixture was
stirred for 1 h, the solvent was evaporated in vacuo. The residue
was purified via silica column chromatography (eluent: Et2O).
The crude product (1.80 g) was recrystallized from PE:CH2Cl2
to give 1.73 g of pure 8b in quantitative yield. Rf 0.30 (PE:EA =
4:1). HPLC (2 ꢀ 150 mm Luna Phenyl-Hexyl 3 μm, acetonitrile:
water, grad: 5 f 95 vol % AN): tR = 23.07 min (>99.0% peak
area). 1H NMR (300 MHz, CD3OD, 20 °C): δ 7.36-7.29 (m, 5 H,
C16-18H), 5.14 (dd, J(H,H) = 12.3 Hz, J(H,H) = 25.1 Hz, 2 H,
C14H2), 4.38 (dd, J(H,H) = 5.6 Hz, J(H,H) = 9.1 Hz, 1 H, C5H
3
C17H2), 5.07 (d, J(H,H) = 7.6 Hz, 1 H, N4H), 4.88 (m, 2 H,
N9H, N11H), 4.57-4.50 (m, 1 H, C15H), 4.34-4.26 (m, 1 H, 1
C7H2), 4.24-4.13 (m, 2 H, C5H, C12H), 4.05-3.93 (m, 2 H, C8H,
1 C7H2), 1.74-1.25 (m, 21 H, 3 C1H3, 4 C22H2, 4 C23H),
0.96-0.85 (m, 24 H, 8 C23H3). 13C NMR (CD3OD): δ 175.9,
174.8, 173.7, 159.7, 158.1, 137.2, 129.6, 129.4, 129.3, 80.5, 68.4,
67.9, 53.5, 53.2, 52.1, 48.1, 42.9, 42.1, 41.6, 41.4, 28.7, 26.0, 25.8,
23.6, 23.39, 23.36, 23.3, 22.5, 21.93, 21.86. HR-ESI-MS: m/z
691.4612 (calcd 691.4646 for C37H62N4O8 þ 1 Hþ).
Boc-(D)-leucine-ester-(L)-leucinol-urea-(D)-leucine-amide-(L)-leu-
cine-Bn (10b). 9b (1.46 g, 3.00 mmol), 1a (0.69 g, 3.11 mmol), and
DMAP (0.37 g, 3.00 mmol) were dissolved in CH2Cl2 (50 mL)
and the mixture was cooled to 0 °C. To the cold solution was
added EDC (0.86 g, 4.50 mmol). The solution was allowed to
warm to room temperature and stirred for 60 h. The reaction
mixture was quenched by the addition of silica gel. After the
mixture was stirred for 4 h, the solvent was evaporated in vacuo.
The solid residue was given on a column and purified via silica
column chromatography (eluent: Et2O). The crude product
(2.20 g) was recrystallized from PE:CH2Cl2 to give 1.98 g
(96%) of pure 10b. Rf 0.10 (PE:EA = 4:1). HPLC (2 ꢀ 150 mm
Luna Phenyl-Hexyl 3 μm, acetonitrile:water, grad: 5 f 95 vol %
3
or C12H), 4.18 (t, J(H,H) = 7.5 Hz, 1 H, C5H or C12H or 1
C7H2), 4.08-4.00 (m, 3 H, 1 C7H2, C8H, 1 C7H2 or C5H or
C12H), 1.78-1.26 (m, 18 H, 3 C1H3, 3 C19H2, 3 C20H),
0.95-0.90 (m, 18 H, 6 C21H3). 13C NMR (CD3OD): δ 174.9,
174.6, 159.8, 158.0, 137.2, 129.5, 129.2, 129.2, 80.3, 68.3, 67.6,
53.5, 52.8, 48.1, 42.3, 41.9, 41.3, 28.8, 25.9, 25.84, 25.77, 23.6,
23.3, 23.3, 22.4, 22.1, 21.8. HR-ESI-MS: m/z 578.3817 (calcd
578.3805 for C31H51N3O7 þ 1 Hþ).
Boc-(L)-leucine-ester-(L)-leucinol-urea-(L)-leucine (9a). To a
solution of 8a (1.73 g, 3.00 mmol) in 50 mL of ethyl acetate
J. Org. Chem. Vol. 75, No. 3, 2010 781

Hartwig et al.
JOCArticle
AN): tR = 23.55 min (>99.0% peak area). 1H NMR (400 MHz,
CD2Cl2, 20 °C): δ7.39-7.29 (m, 5 H, C19-21H), 7.15 (d, 3J(H,H) =
7.8 Hz, 1 H, N14H),5.12(dd, J(H,H) = 12.2 Hz, J(H,H) = 19.5 Hz,
2 H, C17H2), 5.05 (d, 3J(H,H) = 5.6 Hz, 1 H, N4H), 4.92 (d, 3J(H,
H) = 7.3 Hz, 1 H, N9H), 4.82 (d, 3J(H,H) = 9.2 Hz, 1 H, N11H),
4.72-4.66 (m, 1 H, 1 C7H2), 4.62-4.55 (m, 1 H, C15H), 4.20-4.09
(m, 2 H, C5H, C12H), 4.09-4.03 (m, 1 H, C8H), 3.76 (d, J(H,H) =
9.6 Hz, 1 H, 1 C7H2), 1.75-1.21 (m, 21 H, 3 C1H3, 4 C22H2, 4
C23H), 0.97-0.83 (m, 24 H, 8 C23H3). 13C NMR (CD3OD): δ
175.9, 174.9, 173.7, 159.6, 158.2, 137.2, 129.6, 129.3, 129.2, 80.6,
67.9, 67.8, 53.73, 53.68, 52.2, 48.5, 42.9, 41.7, 41.3, 41.2, 28.8, 25.93,
25.87, 23.6, 23.35, 23.31, 22.3, 22.1, 21.9, 21.8. HR-ESI-MS: m/z
691.4651 (calcd 691.4646 for C37H62N4O8 þ 1 Hþ).
Acknowledgment. Generous support by the German
Research Foundation (DFG via SFB 765) and the Fonds
ꢀ
der Chemischen Industrie for providing a doctoral Kekule-
Fellowship to Sebastian Hartwig are gratefully acknow-
ledged. Wacker Chemie AG, BASF AG, Bayer Industry
Services, and Sasol Germany are thanked for generous
donations of chemicals.
Supporting Information Available: Details of building
block and oligomer synthesis as well as compound characteriza-
tion data. This material is available free of charge via the
Internet at http://pubs.acs.org.
782 J. Org. Chem. Vol. 75, No. 3, 2010