Synthesis of a carboxylate functionalized bis-amino acid monomer

Article abstract of DOI:10.1021/jo802679n

(Chemical Equation Presented) The synthesis of the first functionalized bis-amino acid monomer proAc(2S3S4R) 1 that carries an acetyl side chain is presented. This monomer was incorporated into oligomer 3 and the solution phase structure was determined by

Full text of DOI:10.1021/jo802679n

Synthesis of a Carboxylate Functionalized Bis-Amino Acid
Monomer
Sharad Gupta† and Christian E. Schafmeister*,‡
Department of Chemistry, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15260, and
Department of Chemistry, Temple UniVersity, Philadelphia, PennsylVania 19122
meister@pitt.edu
ReceiVed December 5, 2008
The synthesis of the first functionalized bis-amino acid monomer proAc(2S3S4R) 1 that carries an acetyl
side chain is presented. This monomer was incorporated into oligomer 3 and the solution phase structure
was determined by using two-dimensional nuclear magnetic resonance. The solution structure confirmed
the intended connectivity and stereochemistry of the oligomer. This first functionalized bis-amino acid
represents a milestone toward functionalized bis-peptide nanostructures for catalytic, molecular recognition,
and nanotechnology applications.
Introduction
activities and mixed ꢀ-peptide foldamers have been developed
that mimic peptide/protein interactions.5-7 Functionalized pep-
toids have also demonstrated antimicrobial activity and are being
developed as mimics of lung surfactant proteins.8,9 Phenylene
ethynylene foldamers have demonstrated molecular recognition
of small molecules.10 In our own laboratory, we have developed
a functionalized oligomer that acts as a molecular switch under
the control of metal exchange.11 An essential requirement toward
the development of more sophisticated applications for shape-
persistant oligomers is the development of monomers that carry
proteogenic and nonproteogenic chemical functionality.
Oligomers that adopt well-defined three-dimensional struc-
tures have attracted a great deal of interest in the past decade.1-3
Functionalized oligomers that present proteogenic and nonpro-
teogenic functional groups from a structurally defined backbone
have found many applications.4 Functionalized amphipathic
ꢀ-peptides have demonstrated antifungal and antimicrobial
† University of Pittsburgh.
‡ Temple University.
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In our laboratory, we have developed a synthetic approach
to a unique class of oligomers called bis-peptides.12 We
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3652 J. Org. Chem. 2009, 74, 3652–3658
10.1021/jo802679n CCC: $40.75 2009 American Chemical Society
Published on Web 04/24/2009

Synthesis of a Carboxylate Functionalized Bis-Amino Acid Monomer
SCHEME 1. Synthesis of Ketone Intermediates 8a and 8b
FIGURE 1. Previously synthesized bis-amino acid16-18 monomers.
synthesize stereochemically pure, cyclic bis-amino acid mono-
mers and couple them through pairs of amide bonds to create
spiro-ladder oligomers (bis-peptides) with programmable three-
dimensional shapes. Bis-peptides are assembled by using solid
phase peptide synthesis and they are trivially functionalized on
their two ends through the incorporation of amino acids at the
beginning or the end of a bis-peptide synthesis. To enable the
development of more highly functionalized bis-peptides we need
to develop synthetic approaches to incorporate functionality into
the bis-peptide backbone. The approach that we have taken in
this report is to create the functionalized bis-amino acid
“proAc(2S3S4R)” that carries a protected carboxylic acid
functional group on its cyclic core. The carboxylic acid group
is a very versatile proteogenic functional group found in aspartic
acid and glutamic acid. It plays an essential role in enzyme
catalysis such as part of the Asp-His-Ser catalytic triad in
proteases and esterases.13 Miller and co-workers have demon-
strated that carboxylic acids on chiral scaffolds can carry out
asymmetric epoxidation of alkenes.14,15 It is a polar group that
is solvated well by water and it can be activated and further
functionalized through the formation of amides and esters.
to convert 6 into ketone 7 through a three-step procedure that
included the conversion of 6 to its benzyl ester,24,25 protection
of the secondary amine with a phenylfluorenyl group,22 and
oxidation of the alcohol by using Swern oxidation to produce
the ketone 7.26,27 We then treated 7 with n-BuLi in THF:HMPA
at -78 °C and alkylated the resulting enolate using BrCH2CO2t-
Bu at -40 °C. The alkylation occurred exclusively at the
3-position of the ring consistent with previous reports.28 We
obtained a diastereomeric mixture of ketones 8a and 8b in a
10:1 ratio as determined by C18 reverse-phase (RP) HPLC-MS
analysis and 1D proton NMR. After separation of the diaster-
eomers by silica gel chromotography, the stereochemistry of
each diastereomer was tentatively assigned by comparing the
coupling constants between HR and Hꢀ for the respective
ketones (J ) 6.1 Hz for 8a and J ) 8.2 Hz for 8b) with those
reported previously for similar alkylated ketone derivatives
(Scheme 1).28,29 The alkylation of the enolate with BrCH2CO2t-
Bu at -40 °C represents a bottleneck in the overall process of
synthesizing 1. We found that the combined yield of 8a and 8b
decreased significantly when we carried the reaction out on
scales larger than 100 mmol and in particular in round-bottomed
flasks larger than 100 mL. We believe that this is because of
poor temperature control in larger volumes and we are exploring
the use of continuous flow reactors to increase the throughput
of material.
Both diastereomers 8a and 8b could lead to useful carboxylate
functionalized bis-amino acids with different stereochemistry.
In subsequent steps toward the synthesis of these functionalized
bis-amino acids we discovered that intermediate 8a led to
mixtures of diastereomers that we could not separate. On the
other hand, intermediate 8b led to the single, well-behaved bis-
amino acid 1. To increase the amounts of intermediate 8b that
we could produce we carried out a controlled epimerization29
at carbon 3 by treating the 10:1 mixture of 8a and 8b with
lithium bis(trimethylsilyl)amide (LiHMDS) at -78 °C for 1 h
followed by quenching by rapidly adding saturated ammonium
chloride to the reaction mixture at -78 °C. The resulting
diastereomeric mixture has a more useful ratio of 8a and 8b of
1:2. The two diastereomers were then separated by silica gel
chromatography (Scheme 1).
Results and Discussion
Synthesis of the proAc(2S3S4R) monomer starts from trans-
4-hydroxy-L-proline 6sthe same starting material used in the
synthesis of of pro418,19 and pip516 monomers (Figure 1). We
built on the work of Rapoport and Lubell,20-24 who have made
extensive use of the phenylfluorenyl (Pf) group as a nitrogen
protecting group for regio- and stereoselective alkylation of
various amino acid aldehydes and ketone derivatives. The
phenylfluorenyl group offered us several advantages over other
protecting groups (such as carbamates): it simultaneously
masked the secondary amine, protected the stereochemical
configuration of the R-carbon, and protected the adjacent benzyl
ester from attack by nucleophiles and base hydrolysis. The
phenylfluorenyl group was also easily removed by hydrogenoly-
sis and it facilitated NMR analysis because it did not produce
amide rotamers. We used the Rapoport and Lubell chemistry
(12) Schafmeister, C. E.; Brown, Z. Z.; Gupta, S. Acc. Chem. Res. 2008, 41,
1387–1398.
(13) Polga´r, L. Cell. Mol. Life Sci. 2005, 62, 2161.
(14) Jakobsche, C. E.; Peris, G.; Miller, S. J. Angew. Chem., Int. Ed. 2008,
47, 6707–6711.
(15) Peris, G.; Jakobsche, C. E.; Miller, S. J. J. Am. Chem. Soc. 2007, 129,
8710–8711.
(16) Gupta, S.; Das, B. C.; Schafmeister, C. E. Org. Lett. 2005, 7, 2861–
2864.
Toward the synthesis of carboxyl functionalized bis-amino
acid 1 we converted the ketone 8b to a protected amino ester.
(17) Habay, S. A.; Schafmeister, C. E. Org. Lett. 2004, 6, 3369–3371.
(18) Levins, C. G.; Schafmeister, C. E. J. Am. Chem. Soc. 2003, 125, 4702–
4703.
(19) Levins, C. G.; Brown, Z. Z.; Schafmeister, C. E. Org. Lett. 2006, 8,
2807–2810.
(20) Sharma, R.; Lubell, W. D. J. Org. Chem. 1996, 61, 202–209.
(21) Lubell, W. D.; Jamison, T. F.; Rapoport, H. J. Org. Chem. 1990, 55,
3511–3522.
(22) Lubell, W.; Rapoport, H. J. Org. Chem. 1989, 54, 3824–3831.
(23) Lubell, W. D.; Rapoport, H. J. Am. Chem. Soc. 1988, 110, 7447–7455.
(24) Lubell, W. D.; Rapoport, H. J. Am. Chem. Soc. 1987, 109, 236–239.
(25) Webb, T. R.; Eigenbrot, C. J. Org. Chem. 1991, 56, 3009–3016.
(26) Atfani, M.; Lubell, W. D. J. Org. Chem. 1995, 60, 3184–3188.
(27) Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem. 1978, 43, 2480–
2482.
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10860–10863.
(29) Blanco, M. J.; Paleo, M. R.; Penide, C.; Sardina, F. J. J. Org. Chem.
1999, 64, 8786–8793.
J. Org. Chem. Vol. 74, No. 10, 2009 3653

Gupta and Schafmeister
SCHEME 2. Synthesis of ProAc(2S3S4R) Monomer
SCHEME 3. Assembly of a Short Oligomer with
ProAc(2S3S4R)a
a The hydrogens on compound 3 are labeled with the number of the
heavy atom to which they are attached. The hydrogens of methylenes are
labeled “R” if they go into the page and “ꢀ” if they come out.
We achieved this by reducing the ketone 8b to the trichlorom-
ethylcarbinol30 9. The reaction of 8b with trichlormethyl anion
yielded only the single diasteremer 9 as evidenced by RP-HPLC-
MS and 1D proton NMR. Intermediate 9 was then treated with
a basic solution of azide to affect a Jocic rearrangement31 with
reaction conditions similar to those developed by Corey-Link,32
which have been previously used by us17 to synthesize quar-
ternary amino acid derivatives. The Jocic rearrangement pro-
duced the azido acid 10 (Scheme 2).
We incorporated 1 into a heterosequence of three bis-amino
acid monomers using solid phase synthesis protocols that we
have previously developed for synthesizing bis-peptides.16,18 The
oligomeric sequence (L)-Leu-pro4(2S4S)-proAc(2S3S4R)-
pro4(2S4S)-(L)-Tyr was assembled on 2-chlorotritylchloride
resin33 (50 mg scale). The leucine residue was used to enhance
the lipophilicity of the oligomer and the tyrosine residue
provides a UV-chromophore to facilitate purification and
characterization. After the attachment of leucine to the resin,34
each subsequent residue was activated as the 1-hydroxy-7-
azabenzotriazole (-OAt) ester35 for coupling to the growing
oligomer on solid support. Quantitative coupling of the first
pro4(2S4S) monomer was achieved through double coupling
of 2 equiv with respect to the resin loading. The next coupling
of proAc(2S3S4R) to the pro4(2S4S) monomer proved to be
difficult. After screening a range of coupling methods and
reagents we concluded that our general coupling method that
involves activating monomer as the -OAt ester gives the best
results (up to 45% after double coupling with 3 equiv for 4 h).
This may be due to the additional hindrance of the t-Bu protected
acetyl side chain. The subsequent coupling of pro4(2S4S)
monomer to proAc(2S3S4R) monomer was quanititaive. After
coupling tyrosine and removal of the terminal Fmoc group, the
oligomer was cleaved from the resin with trifluoroacetic acid
(TFA)/CH2Cl2. After removal of TFA, the oligomer 13 was
lypholized to remove any traces of TFA (Scheme 3).
The azide 10 was then reduced to the amine by using Zn/
AcOH followed by protection with 9-fluorenylmethyl N-
succinimidyl carbonate (Fmoc-Su) to form 11. The Fmoc
protected amino acid 11 was purified by silica gel chromatog-
raphy and a portion was crystallized from hexanes/ethyl acetate.
The X-ray determined structure revealed that the stereochemistry
was as shown for compound 11 and consistent with our tentative
assignment using NMR. Compound 11 was then treated with
1,3-dicyclohexylcarbodiimide (DCC)/4-(dimethylamino)pyridine
(DMAP) and 2,2,3,3-tetrafluoropropanol to yield ester 12.
Finally the Pf protecting group was exchanged for a Boc
protecting group while simultaneously removing the benzyl
protecting group to unmask the carboxylic acid for coupling.
Use of N,N-diisopropylethylamine (DIPEA) as an additive in
this reaction reduced the reaction time from a week to less than
a day. After final purification we obtained the desired monomer
1 as a white foam in an overall 38% yield from the ketone 8b
(Scheme 2). Only three purification columns are required
through the synthesis from 6. The synthesis we describe is well
optimized and highly reproducible for the scale of 850 mg of
compound 1, which was enough for initial investigations of
compatibility of 1 in solid phase synthesis (for one solid phase
synthesis we need 30 mg of 1, and thus we have enough for 28
couplings). The synthesis of 1 identified that the enolate
alkylation to form 8a and 8b and the trichloromethyl anion
reduction to form 9 are potential bottlenecks that we are working
to scale up.
To form the remaining two diketopiperazine (DKP) rings,
13 was dissolved in an 80 mM solution of ammonium acetate
in 20% acetonitrile/water and heated in an oven at 55 °C. The
reaction progress was monitored by RP-HPLC-MS. After 26 h,
analysis of the reaction mixture revealed a major peak for the
desired product 1 and no trace of 13 or any intermediates. The
crude oligomer was purified by preparative C18 reverse-phase
(30) Dominguez, C.; Ezquerra, J.; Baker, S. R.; Borrelly, S.; Prieto, L.;
Espada, M.; Pedregal, C. Tetrahedron Lett. 1998, 39, 9305.
(33) Barlos, K.; Gatos, D.; Scha¨fer, W. Angew. Chem., Int. Ed. Engl. 1991,
30, 590.
(34) Novabiochem Catalogue 2007.
(31) Jocic, Z. Z. Zh. Russ. Fiz. Khim. OVa. 1897, 29, 97.
(32) Corey, E. J.; Link, J. O. J. Am. Chem. Soc. 1992, 114, 1906–1908.
(35) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
3654 J. Org. Chem. Vol. 74, No. 10, 2009

Synthesis of a Carboxylate Functionalized Bis-Amino Acid Monomer
HPLC and after lypholization, 3 was obtained as a white powder
that we used for NMR studies and high-resolution mass
spectrometry (Scheme 3).
For NMR experiments, the oligomer 3 was dissolved in 350
µL of dimethyl sulfoxide-d6 and 5 µL of trifluoroacteic acid to
protonate all basic groups. We carried out a series of two-
dimensional NMR experiments (COSY, TOCSY, ROSEY,
HMQC, and HMBC) and assigned the hydrogen and carbon
chemical shifts using SPARKY.36 The NMR data confirmed
the expected connectivity and stereochemistry.
We carried out a stochastic conformational37 search in vacuo
using the AMBER9438 force field within the molecular mechan-
ics package MOE.39 The lowest energy conformation is shown
in Figure 2; ROESY correlations between non-J-coupled protons
are superimposed on the structure sorted by intensity, classified
as strong, medium, and weak. The conformational search
revealed two closely spaced lowest energy conformers (separated
by 0.22 kcal/mol) differing in conformation of the pyrolidine
ring A. The ROESY correlations for pyrolidine ring A were
consistent with this in that they did not support a single
conformation.
In the case of pyrolidine ring C, amide proton H18 shows a
strong correlation with H13 but only a medium correlation with
H15R. There is a medium intensity correlation between H15R
and H12. We also observe a medium strength correlation
between H20 and H15ꢀ. These correlations are in agreement
with the structure shown.
The ROESY correlations of pyrrolidine ring E are consistent
with the envelop conformation shownsit is identical with the
envelop conformation that we have previously seen for a
pro4(2S4S) monomer followed by an (S)-tyrosine.18,19 The
amide proton H26 shows a strong correlation with H21ꢀ and a
medium correlation with H23ꢀ. A weak correlation of H20 with
H23R further supports this conformation. We did not observe
ROESY correlations between the tyrosine aromatic hydrogens
and H23R and H23ꢀ as seen in previous structures;18,19 this
may be because this structure was determined in DMSO rather
than water, which would promote a hydrophobic interaction
between the aromatic tyrosine and the methylene 23.
The acetyl side chain appears to prefer a single rotamer
because we observe a 3 Hz (gauche) and 11.5 Hz (anti) J1,3
coupling constant between H13 to H34R and H13 to H34ꢀ. We
cannot absolutely assign the chemical shift of H34R and H34ꢀ
and so there are two possible conformations consistent with these
chemical shifts. The conformation shown in Figure 2 is the lower
energy rotamer by 3.2 kcal/mol.
FIGURE 2. The AMBER94 energy minimized structure of bis-peptide
3 that is most consistent with the superimposed ROESY correlations.
The sequence is (L)-Leu-pro4(2S4S)-proAc(2S3S4R)-pro4(2S4S)-(L)-
Tyr and the pyrrolidine rings are shaded green, red, and green,
respectively. The inset figures are a close-up of the second monomer
(proAc(2S3S4R), red). The colors of the ROESY correlation lines are
color-coded based on their intensity (red ) strong, yellow ) medium,
green ) weak).
In conclusion, we have developed a synthesis for a carboxy-
late functionalized bis-amino acid, proAc(2S3S4R). We dem-
onstrated that it can be incorporated into bis-peptides, deter-
mined the structure of a short bis-peptide oligomer that contains
it, verified its stereochemical configuration, and determined its
conformational preference. The synthesis of this monomer is
general and can be used to incorporate other functional groups.
Experimental Section
General HPLC-MS Procedures. HPLC-MS analysis was
performed on a C18 reverse-phase column (3.5 µm packing, 4.6
mm × 150 mm) coupled to a mass spectrometer (ESI source).
Purification by preparative HPLC was carried out on a semiprep
C18 column (5 µm packing, 19 mm × 100 mm). HPLC mobile
phase: Solvent A “water (0.1% formic acid)”, Solvent B “aceto-
nitrile (0.05% formic acid)”. Method 05_95 indicates that a gradient
was run with initial composition of solvent A 95% and B 5% to
solvent A 5% and B 95% over 30 min. Method 05_100xx indicates
(36) Goddard, T. D.; Kneller, D. G. SPARKY; University of California: San
Francisco, CA, 1989.
(37) Ferguson, D. M.; Raber, D. J. J. Am. Chem. Soc. 1989, 111, 4371.
(38) 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.
(39) MOE, 2005.06 ed.; Chemical Computing Group, Inc.: Montreal, Canada
2005.
J. Org. Chem. Vol. 74, No. 10, 2009 3655

Gupta and Schafmeister
that a gradient was run with initial composition of solvent A 95%
and B 5% to solvent B 100% over 32 min and solvent B 100%
was continued for an additional 8 min. The analytical HPLC-
MS was run at a flow rate of 0.8 mL/min while the preparative
HPLC was run at 18 mL/min of mobile phase.
µL) into HPLC-MS for analysis (method: 05_100xx, UV detection
at 301 nm; 10 tR ) 30.4 min, m/z (ion) 645.2 and 667.2 (M + H+
expected 645.3 and M + Na+ expected 667.3), 9 tR ) 32.7 min).
After completion (ca. 9 h, as indicated by the absence of the peak
corresponding to 9), the reaction was quenched by the addition of
saturated NH4Cl solution (10 mL). The resulting mixture was further
diluted with water (50 mL) and extracted with EtOAc (500 mL).
The organic layer was washed with 1:1 brine/water (50 mL)
followed by brine (25 mL). After drying over anhydrous Na2SO4
for 30 min, the organic layer was decanted and concentrated to
dryness by rotary evaporation under reduced pressure. Any residual
solvent was removed by drying under reduced pressure to yield
crude product 10 as a yellow foam (0.81 g). This crude product
was taken to the next step without any further purification. For
characterization, the above reaction was repeated at 0.30 mmol scale
and the resulting crude product was purified by automated flash
chromatography (50 g silica columns; solvent A: hexanes with 0.1%
AcOH, solvent B: EtOAc with 0.1% AcOH; gradient elution: 0-5
CVs “solvent B 10%” followed by 5-45 CVs “solvent B 10% to
60%”). The desired fractions were pooled together and concentrated
by rotary evaporation. To remove residual acetic acid, the purified
product was redissolved in EtOAc (200 mL) and washed with water
(2 × 20 mL). The organic layer was further washed with brine (20
mL) and after drying over anhydrous sodium sulfate for 20 min,
solvent was removed by rotary evaporation under reduced pressure.
The resulting oil was further dried under reduced pressure overnight
to yield the desired product 10 (172 mg, 0.26 mmol, 87%) as a
white foam.
(2S,3S,4S)-Benzyl 3-((tert-Butoxycarbonyl)methyl)-4-(trichlo-
romethyl)-4-hydroxy-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-
2-carboxylate (9). The pure alkylated ketone 8b (1.06 g, 1.85
mmol) was placed in an oven-dried 50-mL round-bottomed flask
and dissolved in dry THF (18.5 mL) under argon. Dry CHCl3 (9.2
mL) was added and the solution was cooled to -78 °C in an
acetone/dry ice bath. After equilibrating for 30 min, a 1 M solution
of LiHMDS in THF (3.7 mL, 2 equiv) was slowly added to the
reaction mixture via syringe pump (0.5 mL/min). The stirring was
continued for an additional 2 h after which the reaction was
quenched by a quick addition of saturated NH4Cl solution (5 mL,
at room temperature) to the reaction mixture at -78 °C. The
quenched reaction mixture was allowed to warm to room temper-
ature. The resulting suspension was further diluted with water (20
mL) and extracted with EtOAc (250 mL). The organic layer was
washed with 1:1 brine/water (25 mL) followed by brine (25 mL).
After drying over anhydrous Na2SO4 for 30 min, the organic layer
was decanted and concentrated to dryness by rotary evaporation
under reduced pressure. The crude product was purified by
automated Flash Chromatography (3 × 50 g columns connected
in series packed with oven-dried silica; Solvent A: hexanes; Solvent
B: EtOAc; gradient elution: 0-5 CVs “solvent A 100%” followed
by 5-75 CVs “solvent B 0% to 40%”). The fractions were analyzed
by HPLC. On the basis of the analysis, the relevant fractions were
divided into three parts (pure 9, pure 8b, and mixture of 9, 8b, and
8a) and pooled together separately. The three pools were concen-
trated by rotary evaporation and the resulting oils were further dried
under reduced pressure overnight to yield pure product 9 (0.81 g,
1.17 mmol, 63%), recovered ketone 8b (0.15 g, 0.26 mmol, 14%),
and the mixture of 9, 8b, and isomerized ketone 8a (0.17 g, ∼15%),
all as white foams (overall recovery ∼92%). The stereochemistry
for 9 was back-assigned after the determination of the stereochem-
istry at the quaternary center of intermediate 11 by X-ray
crystallography.
1H NMR (500.1 MHz, CDCl3): δ 7.75 (d, J ) 7.6 Hz, 1H),
7.57 (d, J ) 7.4 Hz, 1H), 7.50-7.06 (m, 16H), 4.69 (d, J ) 12.4
Hz, 1H), 4.49 (d, J ) 12.4 Hz, 1H), 3.86 (d, J ) 9.6 Hz, 1H), 3.55
(d, J ) 10.0 Hz, 1H), 3.22 (d, J ) 10.0 Hz, 1H), 2.78 (ddd, J )
10.2, 9.6, 4.0 Hz, 1H), 2.44 (dd, J ) 17.5, 4.0 Hz, 1H), 2.16 (dd,
J ) 17.5, 10.2 Hz, 1H), 1.17 (s, 9H). 13C NMR (125.8 MHz,
CDCl3): δ 173.8, 170.1, 169.7, 145.4, 143.5, 141.6, 140.6, 140.0,
134.5, 129.5, 129.2, 128.9, 128.5, 128.4, 128.3, 128.1, 128.1, 128.0,
127.3, 127.1, 126.9, 120.7, 120.1, 81.5, 76.2, 70.9, 67.5 (CH2), 61.6
(CH), 55.9 (CH2), 46.3 (CH), 32.9 (CH2), 27.8 (CH3, 3C). IR (thin
film): 3061, 3034, 2978, 2936, 2107, 1730, 1451, 1368, 1255, 1157,
HPLC-MS. Method: 05_100xx; UV detection at 301 nm;
9 tR ) 32.8 min, m/z (ion) 692.2 (100%), 694.2 (95.9%) (M + H+
expected 692.2 and 694.2, characteristic for the -CCl3 group). 1H
NMR (500.1 MHz, CDCl3): δ 7.62 (dd, J ) 7.0, 7.0 Hz, 2H),
7.52-7.18 (m, 14H), 7.09 (m, 2H), 5.31 (s, 1H), 4.78 (d, J ) 12.4
Hz, 1H), 4.51 (d, J ) 12.4 Hz, 1H), 3.83 (d, J ) 9.4 Hz, 1H), 3.81
(d, J ) 10.5 Hz, 1H), 3.62 (d, J ) 10.5 Hz, 1H), 3.34 (ddd, J )
9.4, 8.2, 5.0, Hz, 1H), 2.89 (dd, J ) 18.0, 5.0 Hz, 1H), 1.91 (dd,
J ) 18.0, 9.1 Hz, 1H), 1.16 (s, 9H). 13C NMR (125.8 MHz, CDCl3):
δ 176.1, 170.1, 146.3, 146.1, 141.5, 140.6, 140.3, 134.5, 128.9,
128.9, 128.6, 128.6, 128.5, 128.4, 128.3, 128.2, 127.8, 127.6, 127.2,
127.0, 125.9, 120.4, 120.2, 103.1, 88.2, 80.9, 75.5, 67.3 (CH2), 65.2
(CH), 59.5 (CH2), 42.8 (CH), 31.8 (CH2), 27.8 (CH3, 3C). IR (thin
film): 3391, 3062, 2976, 2936, 2877, 1727, 1451, 1368, 1257, 1156,
909, 737. [R]27 +124 (c 0.47, CHCl3). HRMS-ES (m/z): calcd
D
for C38H36N4O6Na+ (M + Na+) 667.2533, found 667.2458.
(2S,3S,4R)-2-((Benzyloxy)carbonyl)-3-((tert-butoxycarbonyl)-
methyl)-4-amino-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-4-car-
boxylic Acid (s1). The crude product 10 obtained in the preceding
step (0.81 g, all of it) was transferred to a 50-mL round-bottomed
flask and dissolved in dry THF (12 mL). AcOH (12 mL) was added
to this solution followed by 0.16 g of Zn dust (20 wt % of 10).
The reaction mixture was stirred at rt and the reaction progress
was monitored by HPLC: a small volume (10 µL) was withdrawn
from the reaction flask, diluted with MeOH (250 µL), and filtered
through Spin-X centrifuge filter. We injected a portion of this
solution (5 µL) into HPLC for analysis (method: 05_100xx, UV
detection at 301 nm; s1 tR ) 20.2 min, m/z (ion) 619.2 and 641.2
(M + H+ expected 619.3 and M + Na+ expected 641.3), 10 tR )
30.4 min). After completion (ca. 3 h, as indicated by the absence
of a peak corresponding to 10) the reaction mixture was filtered
through a filter paper, using a Buchner funnel under reduced
pressure. The residual zinc was washed 2-3 times with a THF/
AcOH (1:1) mixture to extract any trapped product. All the filtrates
were combined and concentrated by rotary evaporation under
reduced pressure. To remove residual acetic acid, the residual solid
was dissolved in CH2Cl2 (5 mL) and diluted with hexanes (20 mL),
then the solvent was removed by rotary evaporation under reduced
pressure. This process was repeated one more time to ensure
complete removal of acetic acid. The resulting glue-like solid was
further dried under reduced pressure overnight to yield crude
product s1 as a white foam (0.75 g) that was taken to the next step
as such. For characterization, a small part of this crude product s1
(35 mg) was dissolved in 1:1 acetonitrile/water (500 µL) and
1065, 909, 794, 735. [R]27 -97.3 (c 0.42, CHCl3). HRMS-ES
D
(m/z): calcd for C38H36Cl3NO5Na+ (M + Na+) 714.1557, found
714.1564.
(2S,3S,4R)-2-((Benzyloxy)carbonyl)-3-((tert-butoxycarbonyl)-
methyl)-4-azido-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-4-car-
boxylic Acid (10). The pure intermediate 9 (0.83 g, 1.2 mmol)
was placed in a 50-mL round-bottomed flask and dissolved in
dioxane (12 mL). The reaction flask was cooled in an ice bath for
2 min and a solution (precooled to 0 °C) of NaN3 (390 mg, 6 mmol,
5 equiv) and NaOH (240 mg, 6 mmol, 5 equiv) in water was added
drop by drop. Additional dioxane (3-4 mL) was added to dissolve
any starting material that precipitates out upon addition of aqueous
solution. The stirring was continued and the reaction progress was
monitored by HPLC: a small volume (5 µL) was withdrawn from
the reaction flask, diluted with MeOH (45 µL), and filtered through
Spin-X centrifuge filter. We injected a portion of this solution (5
3656 J. Org. Chem. Vol. 74, No. 10, 2009

Synthesis of a Carboxylate Functionalized Bis-Amino Acid Monomer
purified by preparative HPLC (method: 05_95, UV detection at
254 nm, tR ) 18.1 min). The products containing fractions from
all three injections were pooled and the solvent was removed by
lyophilization to yield pure product s1 as fluffy white powder (ca.
30 mg), which was used for the characterization tests.
144.1, 143.9, 142.4, 141.9, 141.3, 139.9, 139.7, 134.6, 129.8, 129.3,
129.1, 129.0, 128.8, 128.7, 128.6, 128.3, 128.0, 127.9, 127.7, 127.7,
127.3, 127.2, 127.1, 126.9, 125.5, 125.3, 120.6, 120.0, 82.2, 76.7,
67.2 (CH2), 67.1 (CH2), 65.0, 60.6 (CH), 55.8 (CH2), 47.1 (CH),
42.2 (CH), 33.3 (CH2), 27.9 (CH3, 3C). IR (thin film): 3339, 3063,
3034, 2978, 1728, 1503, 1450, 1256, 1156, 1056, 909, 737. [R]27
1H NMR (500.1 MHz, (CD3)2CO): δ 7.80-7.15 (m, 18H), 4.61
(d, J ) 12.6 Hz, 1H), 4.57 (d, J ) 12.6 Hz, 1H), 3.93 (d, J ) 10.6
Hz, 1H), 3.74 (d, J ) 8.2 Hz, 1H), 3.49 (d, J ) 10.6 Hz, 1H), 2.79
(ddd, J ) 8.8, 8.2, 6.6 Hz, 1H), 2.59 (dd, J ) 18.0, 8.8 Hz, 1H),
2.34 (dd, J ) 18.0, 6.6 Hz, 1H), 1.302 (s, 9H). 13C NMR (125.8
MHz, (CD3)2CO): δ 206.5, 206.3, 206.2, 206.0, 176.1, 172.4, 171.7,
148.5, 147.9, 144.4, 141.4, 141.1, 137.1, 129.6, 129.3, 129.1, 129.1,
129.1, 128.7, 128.5, 128.2, 128.2, 127.9, 126.8, 121.1, 120.7, 81.0,
76.3, 70.3, 66.4 (CH2), 64.1 (CH), 60.3 (CH2), 48.5 (CH), 32.7
(CH2), 28.2 (CH3, 3C). IR (thin film): 3414, 2978, 1720, 1501,
D
+
+143 (c 0.54, CHCl3). HRMS-ES (m/z): calcd for C53H49N2O8
(M + H+) 841.3489, found 841.3497.
(2S,3S,4R)-2-Benzyl 4-(2,2,3,3-Tetrafluoropropyl) 3-((tert-Bu-
toxycarbonyl)methyl)-4-(9H-fluoren-9-yl)methoxycarbonylamino)-
1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-2,4-dicarboxylate (12).
To a solution of 11 (0.88 g, 1.05 mmol) in dry CH2Cl2 (21 mL, 20
mL per mmol) in a 50-mL round-bottomed flask was added DMAP
(13 mg, 0.1 mmol, 0.1 equiv) and 2,2,3,3-tetraflouropropanol (283
µL, 3.15 mmol, 3 equiv). The resulting solution was cooled in an
ice bath under argon. To this cooled solution was added DCC (433
mg, 2.1 mmol, 2 equiv) in one portion. The reaction mixture was
allowed to warm to room temperature and the stirring was continued
overnight. The reaction progress was monitored by HPLC: a small
volume (10 µL) was withdrawn from the reaction flask, diluted
with MeOH (200 µL), and filtered through a Spin-X centrifuge filter.
We injected a portion of this solution (10 µL) into HPLC for
analysis (method: 05_100xx, UV detection at 301 nm; 12 tR ) 34.4
min, 11 tR ) 32.4 min). After completion (ca. 18 h, as indicated
by the absence of the peak corresponding to 11), the reaction was
quenched by the addition of acetic acid (200 µL) and concentrated
to dryness by rotary evaporation. The residual solid was dissolved
in 30% EtOAc/hexanes (100 mL) and filtered through a sintered
funnel to remove the dicyclohexylurea byproduct. The filtrate was
concentrated by rotary evaporation to yield the crude product 12
as a yellow foam. The crude product was purified by automated
flash chromatography (2 × 40 g silica columns connected in series;
solvent A: hexanes, solvent B: EtOAc; gradient elution: 0-5 CVs
“solvent A 100%” followed by 5-45 CVs “solvent B 0% to 30%”).
The desired fractions were pooled and concentrated by rotary
evaporation. The resulting oil was further dried under reduced
pressure overnight to yield 12 as a white foam (0.89 g, 0.93 mmol,
89%).
1256, 1155, 909, 735. [R]27 +19.6 (c 0.28, CHCl3). HRMS-ES
D
(m/z): calcd for C38H39N2O6+ (M + H+) 619.2808, found 619.2825.
(2S,3S,4R)-2-((Benzyloxy)carbonyl)-3-((tert-butoxycarbonyl)-
methyl)-4-((9H-fluoren-9-yl)methoxycarbonylamino)-1-(9-phen-
yl-9H-fluoren-9-yl)pyrrolidine-4-carboxylic Acid (11). The crude
amino acid s1 obtained in the preceding step (0.71 g) was transferred
to a 50-mL round-bottomed flask and dissolved in dioxane (about
12 mL). This was followed by the addition of water (6 mL) and
sodium carbonate (0.25 g, 2.4 mmol, 2 equiv assuming 100%
conversion in the previous 2 steps). More dioxane (5-6 mL) was
added to ensure that any precipitated starting material goes back
into the solution. To this reaction mixture was added Fmoc-Su in
one portion. At this stage the reaction mixture was inhomogeneous.
The reaction progress was monitored by HPLC: a small volume
(10 µL) was withdrawn from the reaction flask, diluted with MeOH
(200 µL), and filtered through a Spin-X centrifuge filter. We injected
a portion of this solution (5 µL) into HPLC for analysis (method:
05_100xx, UV detection at 301 nm; 11 tR ) 32.4 min, m/z (ion)
842.3 (M + H+ expected 842.3), s1 tR ) 22.4 min). After
completion (ca. 17 h, as indicated by the absence of the peak
corresponding to s1), the reaction mixture was quenched by the
dropwise addition of 2 N HCl solution until the solution was at pH
∼4 as monitored by pH paper. The resulting clear solution was
extracted with EtOAc (200 mL + 50 mL). The combined organic
layer was washed with 1:1 brine/water (50 mL) followed by brine
(25 mL). After being dried over anhydrous Na2SO4 for 30 min, the
organic layer was decanted and concentrated by rotary evaporation
under reduced pressure. The crude product was purified by
automated flash chromatography (50 g silica column; solvent A:
hexanes with 0.1% AcOH, solvent B: EtOAc with 0.1% AcOH;
gradient elution: 0-5 CVs “solvent B 10%” followed by 5-55
CVs “solvent B 10% to 60%”). The desired fractions were pooled
together and concentrated by rotary evaporation. To remove the
traces of acetic acid, the residual solid was dissolved in CH2Cl2 (5
mL) and diluted with hexanes (20 mL), then the solvent was
removed by rotary evaporation under reduced pressure. This process
was repeated one more time to ensure the complete removal of
acetic acid. The resulting oil was further dried under reduced
pressure overnight to yield pure product 11 (0.78 g, 0.93 mmol,
81% over three steps, based on 9) as a white foam. A portion of
this product was then recrystallized from ethyl acetates/hexanes to
yield small, colorless granular crystals of 11 which were used for
NMR and other analyses. X-ray structure determination of these
crystals revealed that the stereochemistry of the intermediate 11 is
S at position 3 and R at position 4 (Figure 26 in the Supporting
Information as well as crystallographic data in CIF format).
1H NMR (500.1 MHz, CDCl3): δ 12.470 (br s, 1H), 7.81
(d, J ) 7.6 Hz, 1H), 7.73 (d, J ) 7.3 Hz, 2H), 7.61-7.17 (m,
22H), 7.128 (t, J ) 7.4 Hz, 1H), 6.938 (s, 1H), 4.78 (d, J ) 12.0
Hz, 1H), 4.42 (m, 1H), 4.34 (d, J ) 12.0 Hz, 1H), 4.22 (t, J ) 7.1
Hz, 1H), 4.16 (d, J ) 8.8 Hz, 1H), 4.10 (m, 1H), 3.57 (d, J ) 10.5
Hz, 1H), 3.35 (d, J ) 10.5 Hz, 1H), 3.09 (m, 1H), 2.11 (dd, J )
18.0, 10.5 Hz, 1H), 1.91 (br d, J ) 17.6 Hz, 1H), 1.331 (s, 9H).
13C NMR (125.8 MHz, CDCl3): δ 172.0, 171.2, 170.8, 155.8, 145.2,
1H NMR (500.1 MHz, CDCl3): δ 7.79 (d, J ) 7.6 Hz, 1H),
7.73 (d, J ) 7.6 Hz, 2H), 7.64-7.17 (m, 23H), 7.06 (t, J ) 7.4
Hz, 1H), 6.16 (br t, J ) 52.6 Hz, -CF2CF2H, 1H), 4.81 (d, J )
12.0 Hz, 1H), 4.72-4.64 (m, 1H), 4.59 (d, J ) 9.1 Hz, 1H), 4.43
(d, J ) 12.0 Hz, 1H), 4.35-4.28 (m, 2H), 4.17-4.12 (m, 2H),
3.12 (d, J ) 10.0 Hz, 1H), 3.06 (d, J ) 10.0 Hz, 1H), 2.66-2.61
(m, 1H), 2.34 (dd, J ) 18.3, 11.4 Hz, 1H), 2.05 (br d, J ) 17.5
Hz, 1H), 1.384 (s, 9H). 13C NMR (125.8 MHz, CDCl3): δ 172.9,
169.6, 156.0, 148.0, 144.1, 143.9, 142.3, 141.4, 141.2, 139.3, 135.2,
129.1, 128.9, 128.8, 128.6, 128.6, 127.9, 127.8, 127.5, 127.3, 127.3,
127.1, 127.1, 125.3, 125.2, 120.4, 120.1, 120.0, 109.0 (tt, 1J ) 249
2
Hz, J ) 33 Hz, CH), 82.4, 76.1, 67.2 (CH2), 66.7 (CH2), 64.2,
61.5 (CH), 60.6 (t, 2J ) 30 Hz, CH2), 56.7 (CH2), 47.1 (CH), 44.6
(CH), 32.4 (CH2), 28.0 (CH3, 3C). IR (thin film): 3325, 3061, 3034,
2978, 1741, 1509, 1260, 1164, 909, 737. [R]27 +123 (c 0.37,
D
CHCl3). HRMS-ES (m/z): calcd for C56H50F4N2O8Na+ (M + Na+)
977.3401, found 977.3438.
(2S,3S,4R)-4-((2,2,3,3-Tetrafluoropropoxy)carbonyl)-1-(tert-
butoxycarbonyl)-3-((tert-butoxycarbonyl)methyl)-4-(9H-fluoren-
9-yl)methoxycarbonylamino)pyrrolidine-2-carboxylic Acid (1).
Ester 12 (1.36 g, 1.42 mmol) was transferred to a 100-mL round-
bottomed flask and dissolved in dry THF (28 mL). Solid Boc2O
(1.0 g, 4.3 mmol) was added to this solution followed by careful
addition of 10 wt % Pd/C (136 mg, 10 wt % of 12) in one portion.
The reaction flask was degassed under reduced pressure and
backfilled with H2 gas several times. After 1 h of stirring, a catalytic
amount of DIPEA (25 µL, 10 mol% of 12) was added to speed up
the reaction. The process of degassing and backfilling with H2 gas
was repeated and the stirring was continued overnight. The reaction
progress was monitored by HPLC: a small volume (10 µL) was
withdrawn from the reaction flask, diluted with MeOH (200 µL)
J. Org. Chem. Vol. 74, No. 10, 2009 3657

Gupta and Schafmeister
and filtered through a Spin-X centrifuge filter. We injected a portion
of this solution (10 µL) into HPLC for analysis (method: 05_100xx,
UV detection at 301 nm; 1 tR ) 27.7 min, 12 tR ) 34.4 min). After
completion (ca. 23 h, as indicated by the absence of the peaks
corresponding to 12 or other intermediates), the reaction mixture
was mixed with Celite and the solvent was removed by rotary
evaporation under reduced pressure. The resulting solid mixture
was transferred to a 40 g empty loading column and further dried
overnight under reduced pressure to remove any residual solvent.
This was followed by purification by automated flash chromatog-
raphy (2 × 40 g silica columns connected in series; solvent A:
CH2Cl2 with 0.1% AcOH, solvent B: 5% MeOH in CH2Cl2 with
0.1% AcOH; gradient elution: 0-10 CVs “solvent A 100%”
followed by 10-50 CVs “solvent B 0% to 100%”). The desired
fractions were pooled and concentrated by rotary evaporation. To
remove residual acetic acid, the purified product was dissolved in
EtOAc (200 mL) and washed with water (2 × 20 mL). The
combined organic layer was further washed with brine (20 mL)
and after drying over anhydrous sodium sulfate for 20 min, the
solvent was removed by rotary evaporation under reduced pressure.
The resulting oil was further dried under reduced pressure overnight
to yield 1 as a white foam (0.85 g, 1.17 mmol, 83%).
52.2, 5.1 Hz, -CF2CF2H, 1H), 4.58-4.40 (m, 3H), 4.36-4.30 (m,
2H), 4.21 (t, J ) 6.8 Hz, 1H), 4.10 (d, J ) 11.5 Hz, 1H), 3.68 (d,
J ) 11.5 Hz, 1H), 3.24 (m, 1H), 2.52 (dd, J ) 17.5, 4.7 Hz, 1H),
2.31 (dd, J ) 17.5, 9.6 Hz, 1H), 1.42 (s, 9H), 1.38 (s, 9H). 13C
NMR (125.8 MHz, 20 °C (CD3)2SO): mixture of rotamers δ 171.1
and 170.6, 169.6 and 169.5, 168.7 and 168.6, 155.8, 153.5 and
152.9, 143.7 and 143.5, 140.7, 127.7, 127.1, 125.1, 120.2, 109.0
(t, J ) 249 Hz, CH), 80.4 and 80.3, 79.6, 79.5, 65.9 (CH2), 60.3
and 60.0 (CH), 60.3 (CH2), 53.6 and 53.0 (CH2), 46.5 (CH), 43.5
and 42.8 (CH), 32.2 and 32.1 (CH2), 28.0 and 27.8 (CH3, 3C), 27.7
(CH3, 3C). IR (thin film): 3318, 3061, 3034, 2978, 1742, 1519,
1260, 1156, 909, 737. [R]27 +75.9 (c 0.34, CHCl3). HRMS-ES
D
(m/z): calcd for C35H40F4N2O10Na+ (M + Na+) 747.2517, found
747.2444.
Acknowledgment. This material is based upon work sup-
ported by the NIH/NIGMS (GM067866).
Supporting Information Available: Other experimental
procedures for the synthesis, solid phase assembly of oligomer
3, relevant NMR spectra, and crystallographic data for inter-
mediate 11. This material is available free of charge via the
Internet at http://pubs.acs.org.
1H NMR (500.1 MHz, 77 °C (CD3)2SO): δ 12.32 (br s, 1H),
8.141 (s, 1H), 7.87 (d, J ) 7.6 Hz, 2H), 7.67 (d, J ) 7.6 Hz, 2H),
7.42 (t, J ) 7.6 Hz, 2H), 7.33 (m t, J ) 7.6, Hz, 2H), 6.39 (tt, J )
JO802679N
3658 J. Org. Chem. Vol. 74, No. 10, 2009