The synthesis of curved and linear structures from a minimal set of monomers

Article abstract of DOI:10.1021/jo051639u

Spiro-ladder oligomers of designed shape were assembled from a set of two enantiomeric bis-amino acid monomers. Two tetramers of differing monomer sequence were synthesized to study the effect of monomer stereochemistry upon macromolecular shape. Two-dime

Full text of DOI:10.1021/jo051639u

The Synthesis of Curved and Linear Structures from a Minimal
Set of Monomers
Christopher G. Levins and Christian E. Schafmeister*
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
meister@pitt.edu
Received August 4, 2005
Spiro-ladder oligomers of designed shape were assembled from a set of two enantiomeric bis-amino
acid monomers. Two tetramers of differing monomer sequence were synthesized to study the effect
of monomer stereochemistry upon macromolecular shape. Two-dimensional NMR experiments were
used to determine the conformational preference of the monomers within the context of the
oligomers. The results of this structural study were used to design two pentamers: one resembling
a rod and another with a curved shape. The pentamers were end-labeled with naphthyl and dansyl
groups. The design hypothesis was confirmed by measuring the efficiency of fluorescence resonance
energy transfer between the naphthyl and dansyl fluorophore pair.
Introduction
systematic approach to oligomers with designed tertiary
structures is still elusive15 because of the immense
complexity involved in predicting the folded structure of
molecules with even a few rotatable bonds.16
Our long-term objective is to design, rapidly synthesize,
and study macromolecules that have compact tertiary
A systematic approach to the rapid synthesis of mac-
romolecules with designed shapes and functions would
greatly facilitate the development of biomimetic chem-
istry1 and nanotechnology.2 Oligomer synthesis is an
efficient approach to macromolecules because it is modu-
lar and allows the rapid assembly of large structures from
a collection of small monomers. Many groups are devel-
oping unnatural monomers that are assembled through
single bonds to form oligomers.3-5 Some of these oligo-
mers have strong tendencies to form well-defined second-
ary structures through the influence of weak noncovalent
interactions.6-14 Nevertheless, the development of a
(7) Seebach, D.; Matthews, J. L. Chem. Commun. 1997, 21, 2015-
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Huang, X.; Barchi, J. J., Jr.; Gellman, S. H. Nature 1997, 387, 381-
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1997, 277, 1793-1796.
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Chem. Rev. 2001, 101, 3893-4011.
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Angew. Chem., Int. Ed. 2003, 42, 535-539.
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S. P. A.; Adams, C. L.; Sundaram, A.; Jacobs, J. W.; Schultz, P. G.
Science 1993, 261, 1303-1305.
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Ed. 2002, 41, 325-328.
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S. Z.; Furukawa, M.; Parra, R. D.; Kovalevsky, A. Y.; Mills, J. L.;
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S. L. J. Am. Chem. Soc. 1992, 114, 6568-6570.
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10.1021/jo051639u CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/05/2005
9002
J. Org. Chem. 2005, 70, 9002-9008

Curved and Linear Structures from a Set of Monomers
SCHEME 1. Structures of the pro4(2S4S) 1a and
1b and pro4(2R4R) 2a and 2b Monomers
trans-4-hydroxy-l-proline 3 (Scheme 1). The synthesis of
Fmoc-pro4(2S4S) 1a has been described previously.17 To
access the Fmoc-pro4(2R4R) 2a monomer, we first epimer-
ize trans-4-hydroxy-L-proline 3 at the 2 position in a two-
step process19 to form cis-4-hydroxy-D-proline 4 and then
carry this material through the synthesis developed for
the Fmoc-pro4(2S4S) monomer.17 The benzyl carbamate
(Cbz) protecting group of monomers 1a and 2a can be
converted to the tert-butyl carbamate (Boc) using a one-
pot procedure20 to afford monomers 1b and 2b.
Heterooligomer Synthesis. To investigate the
ability of the Fmoc-pro4(2R4R) monomer 2a to couple
with the Fmoc-pro4(2S4S) monomer 1a we synthesized
two heterosequences, 7 and 8. The synthetic route to 7
is illustrated (Scheme 2). To 100 mg (0.64 mmol/g
loading) of Rink amide resin was coupled Fmoc-(S)-
2-naphthylalanine, followed by the sequence Fmoc-
pro4(2S4S) 1a, Fmoc-pro4(2S4S) 1a, Fmoc-pro4(2R4R)
2a, and Fmoc-pro4(2R4R) 2a. Each coupling was carried
out using double coupling (30 min each) with 2 equiv of
protected amino acid activated with HATU. The oligomer
was cleaved from the resin using trifluoroacetic acid
(TFA) and then subjected to 8% trifluoromethanesulfonic
acid in TFA for 20 min to remove the carboxybenzoyl
protecting group from each monomer producing the
flexible oligomer 6. Compound 6 was then rigidified by
dissolving it in 20% piperidine in dimethylformamide for
2 days at room temperature.21 During this time, the free
secondary amine of the second, third, and fourth mono-
mer attack the methyl ester moieties preceding them in
an intramolecular aminolysis reaction to form a second
amide bond between each adjacent pair of monomers. The
resulting oligomer 7 is a spiro-fused ring system from
atom 5 to atom 30 (Scheme 2). The same process was
carried out using a different sequence of monomers to
create the spiro-fused oligomer 8.
structures and contain small molecule-sized cavities.
Toward this goal, we have developed stereochemically
pure, cyclic, bis-amino acid monomers that couple through
pairs of amide bonds to form spiro-ladder oligomers that
do not fold, but instead display complex shapes by virtue
of their rich stereochemistry and the conformational
preferences of their fused rings.17,18 We have previously
reported monomer 1, named pro4(2S4S), and demon-
strated that it is easily assembled into homooligomers
that form water-soluble molecular rods of controlled
lengths.17 We present the synthesis of monomer 2 that
we have named pro4(2R4R) and demonstrate the syn-
thesis of heterosequences containing both monomers 1
and 2. We also demonstrate that we can create oligomers
that have different three-dimensional shapes by as-
sembling specific sequences of monomers.
Results and Discussion
Monomer Synthesis. The two building blocks Fmoc-
pro4(2S4S) 1a and Fmoc-pro4(2R4R) 2a are synthesized
from the same stereochemically pure starting material,
NMR Structure Characterization. Our goal is to
accurately predict the structures of large oligomers to
SCHEME 2. Synthesis of the Two Heterosequences 7 and 8
J. Org. Chem, Vol. 70, No. 22, 2005 9003

Levins and Schafmeister
enable rational design of macromolecules. Our approach
toward this end is to model large structures based on the
conformational preference of each monomer in the middle
of a trimer. If we call the pro4(2S4S) monomer “A” and
the pro4(2R4R) monomer “B”, then all possible sequences
of three monomers are: AAA, AAB, BAA, BAB (and their
respective enantiomers BBB, BBA, ABB, and ABA). The
conformation of the central monomer of an AAA sequence
was determined in our previous work.17 To determine the
conformation of the central monomer in the context of
the sequences AAB, BAA, and BAB, we determined the
conformation of the monomers within oligomers 7 (se-
quence AABB) and 8 (sequence ABAB) in solution using
two-dimensional nuclear magnetic resonance. In the
ROESY spectrum of oligomer 7, we observe a strong
ROESY correlation between 20H and 15Hâ and a me-
dium strength correlation between 20H and 15HR. In
addition, there is a weak correlation between 13HR and
15HR and no correlation between 13Hâ and 15Hâ. These
data are consistent with the pyrrolidine ring containing
nitrogen 16 existing in an envelope conformation that
avoids a 1,3 interaction between carbon 11 and nitrogen
18, and the diketopiperazine existing in a boat conforma-
tion. This gives us the conformational preference of the
central monomer of an AAB sequence (Figure 1, top). We
also observe a strong ROESY correlation between 28H
and 21Hâ, no observable correlation between 28H and
21HR, a weak correlation between 21HR and 23HR, and
no correlation between 21Hâ and 23Hâ. These observa-
tions are consistent with the pyrrolidine ring containing
nitrogen 24 existing in a conformation that avoids a 1,3
interaction between carbon 19 and nitrogen 26. This
provides the conformational preference of the central
monomer of an ABB sequence (Figure 1, top).
The conformation of the central monomer of a BAB
sequence was determined by examining the ROESY
spectrum of oligomer 8. In the ROESY spectrum of 8,
there was overlap between the correlation peaks of
R-protons 28H and 20H. Although it is not possible to
integrate the peaks directly, by taking a one-dimensional
slice through the correlation peaks of 20H we were able
to determine that the cross-peak between 20H and 15Hâ
is stronger than the cross-peak between 20H and 15HR.
In addition, there is a weak cross-peak between 13HR
and 15HR and no observed correlation between 13Hâ and
15Hâ. These observations suggest that the second pyr-
rolidine ring, containing atoms 12 through 16, is in an
envelope conformation that avoids a 1,3 interaction
between atoms 11 and 18 (Figure 1, bottom).
Design and Synthesis of Shaped Oligomers. On
the basis of the conformational preferences determined
above, we created two sequences designed to form a
molecular rod 10 (an AAAAA sequence) and a curved
shape 12 (an ABABA sequence) and used fluorescence
resonance energy transfer to test our design. Intermedi-
FIGURE 1. Stereo images of the modeled structures of
oligomers 7 (top) and 8 (bottom). The ROESY correlations are
superimposed in color (red: strong correlation, yellow: me-
dium, green: weak, and gray: intensity is unassigned).
Methylene hydrogens are labeled R if they are below the plane
of the page and â if they are above the plane of the page as
drawn in Scheme 2.
ates 9 and 11 were synthesized using methodology
similar to that described for the synthesis of 7 and 8, with
the exception that Boc protected monomers 1b and 2b
were utilized because we observed decomposition of the
dansyl labeled scaffolds with the conditions that we use
for Cbz group removal. Oligomers 10 and 12 were
expected to form when intermediates 9 and 11 were
dissolved in a solution of 20% piperidine in dimethyl-
formamide for 2 days (Scheme 3).21 In dimethylform-
amide alone, the rate of diketopiperazine formation is
undetectable. In 20% piperidine/DMF at room tempera-
ture, the half-life of diketopiperazine formation is ap-
proximately 1 h. Diketopiperazines are known to epimer-
ize under basic conditions,22 and we were concerned that
upon formation, the diketopiperazine rings within these
longer oligomers might epimerize in the 20% piperidine
solution. To partially address this question, we monitored
the diketopiperazine formation as a function of time by
reverse-phase high-performance liquid chromatography
(17) Levins, C. G.; Schafmeister, C. E. J. Am. Chem. Soc. 2003, 125,
4702-4703.
(18) Habay, S. A.; Schafmeister, C. E. Org. Lett. 2004, 6, 3369-
3371.
(19) Baker, G. L.; Fritschel, S. J.; Stille, J. R.; Stille, J. K. J. Org.
Chem. 1981, 46, 2954-2960.
(20) Sakaitani, M.; Hori, K.; Ohfune, Y. Tetrahedron Lett. 1988, 29,
2983-2984.
(21) Pedroso, E.; Grandas, A.; Delasheras, X.; Eritja, R.; Giralt, E.
Tetrahedron Lett. 1986, 27, 743-746.
(22) Gund, P.; Veber, D. F. J. Am. Chem. Soc. 1979, 101, 1885-
1887.
9004 J. Org. Chem., Vol. 70, No. 22, 2005

Curved and Linear Structures from a Set of Monomers
SCHEME 3. Rigidification of Oligomers 10 and 12
(HPLC) with mass spectrometry (Figure 2). After 3 h, a
large number of intermediates containing less than five
diketopiperazines were observed (Figure 2B). After 32 h,
the reaction is essentially complete; the intermediates
have disappeared and only the product 10 remains
(Figure 2C). Two small peaks with later retention times
and with the identical mass of compound 10 are visible;
these minor impurities are likely epimers of compound
10. Oligomer 10 was cleanly purified by preparative
reverse-phase HPLC.
Fluorescence Measurements. Oligomer 10 was de-
signed to form a molecular rod about 20 Å long, and
curved oligomer 12 was designed to hold its two ends
closer together. Modeling compound 10 using the
Amber8923 and Amber9424 force fields predicts that the
distance from the pyrrolidine nitrogen of the first mono-
mer to the quaternary center of the last monomer will
be 19 and 21 Å respectively. Modeling compound 12 using
the Amber89 and Amber94 force fields predicts that the
distance from the pyrrolidine nitrogen of the first mono-
mer to the quaternary center of the last monomer will
be 12 and 16 Å, respectively. Both oligomers were labeled
on one end with a naphthylalanine fluorescent donor and
FIGURE 2. Progress of diketopiperazine formation as moni-
tored by C18 reverse-phase HPLC, 5% to 95% acetonitrile, 30
min, 0.1% TFA.
(23) Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio,
C.; Alagona, G.; Profeta, S.; Weiner, P. J. Am. Chem. Soc. 1984, 106,
765-784.
(24) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K.
M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.;
Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179-5197.
on the other end with a dansyl acceptor. We also
synthesized two controls: compound 13, wherein the
J. Org. Chem, Vol. 70, No. 22, 2005 9005

Levins and Schafmeister
FIGURE 3. Excitation spectra of four dansylated molecules 10, 12, 13, and 14. The structures are inset, and their colors correspond
to the color of their respective excitation spectrum.
dansyl group is directly connected to the naphthylalanine
donor, and compound 14, labeled only with the dansyl
group. The excitation spectra of all four molecules are
shown (Figure 3). Strong 520-nm emission results from
293-nm excitation when the dansyl and naphthyl groups
are in close proximity, as resonance energy transfer
increases with decreasing donor-acceptor distance.25 The
excitation spectrum of 14 (black) is that of a dansyl group
infinitely far away from a naphthyl donor. The excitation
spectrum of 13 (green) demonstrates the strong ab-
sorbance by the naphthyl group and highly efficient
resonance energy transfer to the dansyl group when they
are coupled through a short linker. The spectrum of 10
(blue), the oligomer designed to form the rod, demon-
strates resonance energy transfer that is ∼50% efficient
(at 293 nm), which is reasonable considering that the
Fo¨rster distance of the dansyl/naphthyl pair is 22 Å.26
The excitation spectrum of the curved oligomer 12 shows
resonance energy transfer at 293-nm excitation that is
about 72% efficient, which suggests that 12 has a curved
shape, as designed. Assuming that the angular relation-
ship between the dansyl and naphthyl groups is sub-
stantially randomized during the excited-state lifetime,25
the calculated distances between the donor acceptor pair
are 22 and 18.5 Å for the rod-shaped compound 10 and
the curved compound 12, respectively. These values are
more consistent with the predictions of the Amber94 force
field. However, recent single-molecule fluorescence mea-
surements reveal that distances measured using fluo-
rescence resonance energy transfer are overestimated
when the donor acceptor pair are closer to each other
than the Fo¨rster distance.27 This suggests that the curved
oligomer 12 may hold its donor acceptor pair closer
together than 18.5 Å. Interestingly, the excitation spectra
(not shown) of the flexible precursors 9 and 11 (Scheme
3) are indistinguishable from one another and nearly
identical to the excitation spectrum of the curved com-
pound 12. An explanation for this is that prior to
rigidification of 10 and 12 the flexible oligomers 9 and
11 exist as an ensemble of conformations where the
dansyl and naphthyl groups are close enough to one
another to permit efficient resonance energy transfer. It
is only after the molecules are rigidified that they exhibit
the behavior inherent to their design.
Conclusion
We have demonstrated the synthesis of the enantio-
meric bis-amino acid monomer pro4(2R4R) 2 and that
we can construct heterosequences of a minimal set of two
monomers, 1 and 2. We have further demonstrated that
the three-dimensional structure and physical properties
of our bis-amino acid containing oligomers can be con-
trolled by the sequence of monomers from which they are
composed.
Experimental Section
Compound 2a. Compound sc8 (shown in Supporting
Information, 4.1 g, 6.8 mmol) was transferred to a 200-mL
round-bottom flask with a magnetic stir bar and dissolved in
(25) Stryer, L.; Haugland, R. P. Proc. Natl. Acad. Sci. U.S.A. 1967,
58, 719-726.
(26) Wu, P.; Brand, L. Anal. Biochem. 1994, 218, 1-13.
(27) Schuler, B.; Lipman, E. A.; Steinbach, P. J.; Kumke, M.; Eaton,
W. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2754-2759.
9006 J. Org. Chem., Vol. 70, No. 22, 2005

Curved and Linear Structures from a Set of Monomers
a solution of 1:1 TFA/CH2Cl2 (70 mL). The reaction mixture
was stirred for 4 h and then concentrated by rotary evapora-
tion; the resulting oily residue was dissolved in toluene and
concentrated again. Solvent was removed under vacuum
overnight, yielding 2a (3.4 g, 6.4 mmol, 94%) as a foam, used
without further purification. An analytical sample was purified
by chromatography on silica (gradient elution over 14 column
volumes from CHCl3 (0.1% AcOH) to 95:5 CHCl3/MeOH (0.1%
(∼1 atm). The reaction mixture was filtered, and the filtrate
was concentrated by rotary evaporation to an oil, which was
purified by chromatography on silica (gradient elution over
10 column volumes from CHCl3 to 10% MeOH/CHCl3). Frac-
tions containing the desired product were concentrated, and
residual solvent was removed under reduced pressure to give
1b (4.5 g, 8.8 mmol, 83% from sc9) as a white foam. 1H NMR
(300 MHz, DMSO-d6): mixture of rotamers δ 8.32 (s, Fmoc-
NH-, 1H), 7.93 (d, J ) 7.4 Hz, 2H), 7.75 (d, J ) 7.1 Hz, 2H),
7.48-7.35 (m, 4H), 4.29-3.94 (m, 5H), 3.58 (s, 3H), 3.41-3.32
(m, 1H), 2.76-2.59 (m, 1H), 2.18-2.08 (m, 1H), 1.39 and 1.34
(s, 9H, rotameric); 13C NMR (75.4 MHz, DMSO-d6): mixture
of rotamers δ 174.9, 172.4, 155.6, 153.4 and 153.3, 143.6 and
143.5, 140.6, 127.5, 127.0, 125.1, 120.0, 78.7 and 78.5, 65.6
(CH2), 62.6 and 61.9, 59.1 and 58.9 (CH), 55.0 and 54.5 (CH2),
52.3 (CH3), 46.5 (CH), 39.3 and 38.4 (CH2), 28.0 and 27.9 (CH3,
3C); IR (neat film) 3307, 3016, 2979, 1744, 1529, 1477, 1450,
1408, 1369, 1254, 1156, 1088, 1050, 972, 913, 858, 759, 667
cm-1; [R]D -61.8° (c 1.42, CHCl3); ESI-MS m/z (relative
intensity): 533.2 (100%, M + Na+), 477.1 (35%), 433.1 (90%),
411.2 (20%); HRESIQTOFMS calcd for C27H30N2NaO8 (M +
Na+) 533.1900, found 533.1887.
Compound 7. A quantity of 100 mg of Rink Amide AM
resin (0.63 mmol/g substitution) was transferred to a solid-
phase reaction vessel and swollen in DMF for several hours.
The N-Fmoc protecting group of the resin was removed by
treatment with a solution of 20% piperidine/DMF for 40 min.
The resin was washed sequentially with DMF, CH2Cl2, i-PrOH,
and DMF. (S)-N-Fmoc-2-naphthylalanine (56.4 mg, 128 µmol)
and HATU (48.6 mg, 128 µmol) were added to a polypropylene
microcentrifuge tube. The protected amino acid and coupling
reagent were dissolved in 640 µL of 20% CH2Cl2/DMF. DIPEA
(45.0 µL, 256 µmol) was added to the coupling solution; the
solution was mixed and added to the resin immediately. The
resin and coupling solution were agitated for 1 h and then
washed. The coupling reaction was repeated one additional
time. The resin was capped with a solution of 400:100:8 DMF/
Ac2O/DIPEA. The naphthylalanine N-terminal Fmoc protect-
ing group was removed using a solution of 20% piperidine/
DMF over 40 min, and the resin was washed sequentially with
DMF, CH2Cl2, i-PrOH, and DMF.
Monomer 1a (70.0 mg, 128 µmol) was coupled to the resin
using HATU in a similar fashion. After the attachment of the
first monomer to the solid phase, the resin was capped, the
N-terminal Fmoc protecting group was removed, and the resin
was washed as described above. The monomer coupling
procedure was repeated three additional times using the
appropriate monomers (1a, 2a, then 2a) and the final N-
terminal Fmoc protecting group was removed. The resin was
then washed with CH2Cl2, MeOH, and CH2Cl2 and then dried
under reduced pressure overnight.
The dried resin was transferred to a 4-mL conical vial
containing a magnetic spin vane. Triisopropylsilane (86 µL),
water (86 µL), and TFA (3.25 mL) were added sequentially.
This cleavage solution was mixed for 2 h, at which time the
resin was filtered from the solution and washed with an
additional volume of TFA. The filtrates were combined and
concentrated by centrifugal evaporation at room temperature.
Thioanisole (25 µL) and ethanedithiol (10 µL) were added to
the resulting oily residue, followed by TFA (250 µL) and triflic
acid (25 µL). This solution was stirred for 15 min and then
added slowly to diethyl ether (∼80 mL); the precipitate (crude
compound 6) that evolved was pelleted by centrifugation. The
ether was decanted from the pellet, and the pellet was
dissolved in 20% piperidine/DMF (1.25 mL). After approxi-
mately 48 h at room temperature, the crude product was
precipitated from ether. The precipitate was dissolved in a 90:
10:1 H2O/MeCN/TFA solution, and the product was purified
by preparative HPLC (C18 column; 30 mm × 300 mm; mobile
phase, MeCN (0.05% TFA)/water (0.1% TFA), 10% to 35%
MeCN over 30 min; flow rate, 43 mL/min). The fractions
containing the desired product were concentrated by lyo-
1
AcOH)). H NMR (300 MHz, 75 °C, DMSO-d6): δ 7.96 (br s,
Fmoc-NH-, 1H), 7.86 (d, J ) 9.3 Hz, 2H), 7.68 (d, J ) 7.5 Hz,
2H), 7.43-7.31 (m, 9H), 5.11 (s, 2H), 4.36 (m, 3H), 4.22
(apparent t, J ) 6.3 Hz, 1H), 4.08 (d, J ) 9.0 Hz, 1H), 3.61 (s,
3H), 3.58 (overlap with -CO2CH3, 1H), 2.84 (m, 1H), 2.28 (dd,
J ) 12.8, 5.6 Hz, 1H); 13C NMR (75.4 MHz, DMSO-d6):
mixture of rotamers δ 172.5, 172.2, 171.9, 155.6, 153.6 and
153.3, 143.5 and 143.4, 140.6, 136.5, 128.2, 128.1, 127.7, 127.4,
127.3, 126.9, 125.0, 119.9, 66.1 (CH2), 65.6 (CH2), 62.7 and 61.9,
57.7 and 57.4 (CH), 54.8 and 54.4 (CH2), 52.5 (CH), 46.5 (CH3),
37.6 and 37.5 (CH2); IR (neat film) 3308, 3065, 2953, 1715,
1529, 1450, 1423, 1357, 1251, 1110, 1085, 1051, 973, 826, 760,
740, 699 cm-1; [R]D +31.3° (c 1.4, CHCl3); HPLC: C18 column,
mobile phase, MeCN (0.05% TFA)/water (0.1% TFA), 5% to
95% MeCN over 30 min, 1.00 mL/min, UV detection at 274
nm; tR for 2a, 23.80 min; ESI-MS m/z (relative intensity): 567.2
(100%), 501.2 (11%); HRESIQTOFMS calcd for C30H28N2NaO8
(M + Na+) 567.1743, found 567.1743.
Compound 2b. A 500-mL round-bottom flask was charged
with a magnetic stir bar, 2a (3.4 g, 6.4 mmol), and 10 wt %
Pd/C (679 mg). THF (190 mL) was added to the flask, and the
reaction mixture was degassed through repeated cycles of
evacuation under reduced pressure followed by backfilling with
H2. The solution was stirred under a H2 atmosphere (∼1 atm)
overnight. The Pd/C was filtered from the solution and washed
with EtOAc. The filtrates were concentrated by rotary evapo-
ration. The resulting oil was purified by chromatography on
silica (gradient elution over 10 column volumes from CHCl3
to 10% MeOH/CHCl3). Fractions containing the desired prod-
uct were combined and concentrated by rotary evaporation and
then under reduced pressure overnight yielding 2b (2.7 g, 5.4
mmol, 80% from sc8) as a white foam. 1H NMR (300 MHz,
DMSO-d6): mixture of rotamers δ 8.29 (s, Fmoc-NH-, 1H), 7.88
(d, J ) 7.4, 2H), 7.71 (d, J ) 7.1, 2H), 7.44-7.31 (m, 4H), 4.31-
4.20 (m, 4H, rotameric), 3.98 (apparent dd, J ) 24.3, 11.2 Hz,
1H), 3.60 (s, 3H), 3.48 (apparent t, J ) 10.4 Hz, 1H), 2.89-
2.76 (m, 1H), 2.29-2.20 (m, 1H), 1.41 and 1.36 (s, 9H,
rotameric); 13C NMR (75.4 MHz, DMSO-d6): mixture of
rotamers δ 173.5, 172.2, 155.7, 153.3, 152.9, 143.6 and 143.5,
140.7, 127.6, 127.0, 125.2, 120.0, 79.0, 65.6 (CH2), 62.7 and
61.9, 58.1 and 57.7 (CH), 54.8 and 54.4 (CH2), 52.5 (CH3), 46.6
(CH), 38.8 and 37.9 (CH2), 28.0 and 27.8 (CH3, 3C); IR (neat
film) 3307, 3016, 2979, 1744, 1530, 1477, 1450, 1409, 1368,
1297, 1253, 1217, 1160, 1088, 1050, 972, 913, 858, 759, 667
cm-1; [R]D +61.8° (c 1.9, CHCl3); HPLC: C18 column, mobile
phase, MeCN (0.05% TFA)/water (0.1% TFA), 5% to 95%
MeCN over 30 min, 1.00 mL/min, UV detection at 274 nm, tR
for 2b, 23.20 min.; ESI-MS m/z (relative intensity): 533.2
(100%, M + Na+), 477.1 (30%), 433.1 (90%); HRESIQTOFMS
calcd for C27H30N2NaO8 (M + Na+) 533.1900, found 533.1904.
Compound 1b. Compound sc9 (shown in Supporting
Information, 6.30 g, 11.5 mmol) was prepared as described
previously,17 transferred to a 500-mL round-bottom flask
containing a magnetic stir bar, and dissolved in 1:1 CH2Cl2/
TFA (210 mL). The reaction mixture was stirred at room
temperature and monitored by TLC (5:1 CHCl3/MeOH, start-
ing material sc9 Rf 0.7, 1b Rf 0.4). When all of the starting
material had been consumed (approximately 4 h), the solution
was concentrated by rotary evaporation, and residual solvent
was removed under reduced pressure overnight. Pd/C (10 wt
%, 1.1 g), THF (300 mL), and Boc2O (7.2 mL, 32 mmol) were
added to the flask, and the solution was degassed under
reduced pressure and then back-filled with H2 gas. The
mixture was stirred overnight under a hydrogen atmosphere
J. Org. Chem, Vol. 70, No. 22, 2005 9007

Levins and Schafmeister
philization, yielding 7 (10.3 mg, 12.9 µmol, ∼20% overall yield,
based upon initial resin loading): HRESIQTOFMS calcd for
C38H43N10O10 (M + H+) 799.3158, found 799.3251.
evaporation, yielding compound 11: HRESIQTOFMS calcd for
C61H77N14O17S (M + H+) 1309.5312, found 1309.5237.
This sample of 11 was dissolved in UV/vis grade MeOH (1.5
mL) and filtered through a 0.2-µm Nylon frit. A sample for
fluorescence spectroscopy was prepared by diluting an aliquot
(7.4 µL) of this stock solution into MeOH (3 mL).
Compound 12. The remaining larger portion of the residue
that had afforded 9 was dissolved in 20% piperidine/DMF (1.25
mL) and transferred to a sealed amber HPLC vial. After ∼48
h, the solution was added slowly to diethyl ether (45 mL); the
resulting precipitate was pelleted by centrifugation. After
decanting the ether, the pellet was dissolved in a 50:50:1 H2O/
MeCN/TFA, and the desired product was purified by prepara-
tive HPLC using the standard method. The desired fractions
were pooled and concentrated to dryness by centrifugal
evaporation, affording compound 12: HRESIQTOFMS calcd
for C57H61N14O13S (M + H+) 1181.4263, found 1181.4182.
Compound 8. 8 was prepared following a procedure similar
to the one described for the synthesis of 7. (S)-N-Fmoc-2-
naphthylalanine, 1a, 2a, 1a, and 2a were coupled sequentially
to 100 mg of Rink Amide AM resin. The resin was cleaved
using TFA, and the Cbz groups of the resulting crude product
were removed with exposure to triflic acid. The product was
treated with 20% piperidine/DMF for 48 h, precipitated from
ether, and purified by preparative HPLC. Fractions containing
the desired product were concentrated by lyophilization,
yielding 8 (10.4 mg, 13.0 µmol, ∼20% overall yield, based upon
initial resin loading): HRESIQTOFMS calcd for C38H43N10O10
(M + H+) 799.3158, found 799.3190.
Compound 9. A quantity of 10 mg of Dansyl NovaTag resin
(0.51 mmol/g substitution) was transferred to a solid-phase
reactor and swollen in DMF. The N-terminal Fmoc protecting
group was removed by mixing the resin with 20% piperidine/
DMF for 40 min. The resin was then washed with DMF,
CH2Cl2, i-PrOH, CH2Cl2, and DMF. 1b, 1b, 1b, 1b, 1b, and
(S)-N-Fmoc-1-naphthylalanine were coupled sequentially to
the resin using the method described for the synthesis of
compound 7; each coupling to the resin (2 equiv of N-Fmoc
protected amino acid, 2 equiv of HATU, 4 equiv of DIPEA, 0.2
M in 20% CH2Cl2/DMF, 30 min reaction time) was repeated
an additional time and was followed by washing, resin capping
with a 400:100:8 DMF/Ac2O/DIPEA solution, and deprotection
of the terminal N-Fmoc group with 20% piperidine/DMF. After
careful washing, residual solvent was removed from the resin
overnight under reduced pressure. The resin was cleaved using
95% TFA, 2.5% triisopropylsilane, and 2.5% water over 2 h
with stirring. The cleavage solution was filtered, the resin was
washed with an additional volume of TFA, and the filtrates
were combined and concentrated by centrifugal evaporation.
One-quarter of the resulting residue was purified by prepara-
tive HPLC (C18 column; mobile phase, MeCN (0.05% TFA)/
H2O (0.1% TFA), 5% to 95% MeCN over 30 min; flow rate, 15
mL/min, herein, the “standard method”). Fractions contain-
ing the desired product were pooled and concentrated to
dryness by centrifugal evaporation, yielding compound 9:
HRESIQTOFMS calcd for C61H77N14O17S (M + H+) 1309.5312,
found 1309.5281.
This sample of 12 was dissolved in UV/vis grade MeOH (1.5
mL) and filtered through a 0.2-µm Nylon frit. A sample for
fluorescence spectroscopy was prepared by diluting an aliquot
(150 µL) of this stock solution into MeOH (3 mL).
Compound 13. (S)-N-Fmoc-1-naphthylalanine was coupled
to Dansyl NovaTag resin (10 mg) using the method described
above. Following removal of the N-Fmoc group of the naph-
thylalanine, resin cleavage, and filtration of the resin, the
cleavage solution was concentrated by centrifugal evaporation.
The resulting residue was dissolved in 40:60:0.1 MeCN/H2O/
TFA (1 mL) and purified by preparative HPLC by the standard
method. The desired fractions were pooled and concentrated
to dryness by centrifugal evaporation, giving compound 13:
HRESIQTOFMS calcd for C27H31N4O3S (M + H+) 491.2117,
found 491.2092.
This sample of 13 was dissolved in UV/vis grade MeOH (1.5
mL) and filtered through a 0.2-µm Nylon frit. A sample for
fluorescence spectroscopy was prepared by diluting an aliquot
(2.5 µL) of the stock solution into MeOH (3 mL).
Compound 14. 1b and (S)-N-Fmoc-leucine was coupled to
Dansyl NovaTag resin (10 mg) sequentially using the method
described above. After removal of the N-Fmoc group of the
leucine, resin cleavage, and filtration of the resin, the cleavage
solution was concentrated by centrifugal evaporation. The
resulting residue was dissolved in 40:60:0.1 MeCN/H2O/TFA
(1 mL) and purified by preparative HPLC by the standard
method. The desired fractions were pooled and concentrated
to dryness by centrifugal evaporation, giving compound 14:
HRESIQTOFMS calcd for C26H37N6O5S (M + H+) 545.2546,
found 545.2516.
This sample of 9 was dissolved in UV/vis grade MeOH (1.5
mL) and filtered through a 0.2-µm Nylon frit. A sample for
fluorescence spectroscopy was prepared by diluting an aliquot
(22.6 µL) of this stock solution into MeOH (3 mL).
Compound 10. The remaining larger portion of the residue
that had afforded 9 was dissolved in 20% piperidine/DMF (1.25
mL) and transferred to a sealed amber HPLC vial. Diketo-
piperazine closure was monitored by analytical HPLC-MS.
After ∼48 h, the solution was added slowly to diethyl ether
(45 mL); the resulting precipitate was pelleted by centrifuga-
tion. After decanting the ether, the pellet was dissolved in a
50:50:1 H2O/MeCN/TFA, and the desired product was purified
by preparative HPLC using the standard method. The desired
fractions were pooled and concentrated to dryness by centrifu-
gal evaporation, affording compound 10: HRESIQTOFMS
calcd for C57H61N14O13S (M + H+) 1181.4263, found 1181.4136.
This sample of 10 was dissolved in UV/vis grade MeOH (1.5
mL) and filtered through a 0.2-µm Nylon frit. A sample for
fluorescence spectroscopy was prepared by diluting an aliquot
(10.0 µL) of this stock solution into MeOH (3 mL).
Compound 11. 1b, 2b, 1b, 2b, 1b and (S)-N-Fmoc-1-
naphthylalanine were coupled sequentially to 10 mg of Dansyl
NovaTag resin using the method described for the synthesis
of compound 9. Following removal of the terminal N-Fmoc
group, resin cleavage, filtration of the resin, and concentration
of the filtrates by centrifugal evaporation, one-quarter of the
resulting residue was purified by preparative HPLC using the
standard method. Fractions containing the desired product
were pooled and concentrated to dryness by centrifugal
This sample of 14 was dissolved in UV/vis grade MeOH (1.5
mL) and filtered through a 0.2-µm Nylon frit. A sample for
fluorescence spectroscopy was prepared by diluting an aliquot
(15 µL) of this stock solution into MeOH (3 mL).
Acknowledgment. We are very thankful for finan-
cial support from the National Institutes of Health/
NIGMS (GM067866). We also thank Dr. Stephane
Petoud for assistance with the fluorescence spectroscopy
and Dr. David Waldeck for helpful discussions.
Supporting Information Available: General procedures,
experimental details for synthesis of monomers 1b, 2a, and
2b, and characterization of new compounds. 1H and 13C spectra
of intermediates in synthesis of monomers. HPLC-MS chro-
matograms of compounds 7, 8, 9, 10, 11, 12, 13, and 14.
Excitation spectra of compounds 9 and 11. Two-dimensional
NMR spectra for compounds 7 and 8. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO051639U
9008 J. Org. Chem., Vol. 70, No. 22, 2005