Synthesis of a PNA-encoded cysteine protease inhibitor library

Article abstract of DOI:10.1016/j.tet.2004.05.107

Peptide nucleic acids (PNAs) have been used to encode a combinatorial library whereby each compound is labeled with a PNA tag which reflects its synthetic history and localizes the compound upon hybridization to an oligonucleotide array. We report herein

Full text of DOI:10.1016/j.tet.2004.05.107

Tetrahedron 60 (2004) 8677–8690
Synthesis of a PNA-encoded cysteine protease inhibitor libraryq
Franc¸ois Debaene,a Lorenzo Mejias,a Jennifer L. Harrisb,c and Nicolas Winssingera
,
*
a
´
´
´
Institut de Science et Ingenierie Supramoleculaires, Universite Louis Pasteur, 8 allee Gaspard Monge, 67000 Strasbourg, France
bDepartment of Chemistry, Genomics Institute of the Novartis Research Foundation, 10675 J. J. Hopkins Dr., San Diego, CA 92121, USA
cDepartment of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
Received 12 November 2003; accepted 4 May 2004
Available online 29 July 2004
Abstract—Peptide nucleic acids (PNAs) have been used to encode a combinatorial library whereby each compound is labeled with a PNA
tag which reflects its synthetic history and localizes the compound upon hybridization to an oligonucleotide array. We report herein the full
synthetic details for a 4000 member PNA-encoded library targeted towards cysteine protease.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
no redundant library members. Furthermore, the library can
be screened in solution prior to hybridization which should
reduce nonspecific interactions and allows the separation of
bound and unbound ligand by size exclusion. This latter
point is important since, by including a label with the PNA
tag, the isolated ligands can be identified directly upon
hybridization thereby avoiding the necessity to label the
target.8
Microarray based technologies have proven to be a powerful
analytical tool for their ability to consider large numbers of
data points in a miniaturized format. Such microarrays can
be prepared by several techniques including photolitho-
graphy, contact printing and inkjet to generate arrays with
densities ranging from 1000 to 500,000 features per square
centimeters.1 The success of microarrays in genomic
analysis2 has prompted researchers in other areas to adopt
this format. To date, a number of chemistries have been
developed to derivatize glass surfaces for protein,3 oligo-
saccharide4 and small molecule microarrays.5 A strong
motivation for developing small molecule microarrays is to
miniaturize high throughput screening. Considering that an
arrayed glass slide prepared by contact printing can present
10,000 analytes to be screened in 50 ml, this represent a
1000 fold miniaturization over the 1536 microtiter plate
format. Another application of small molecule microarrays
is to measure the functional activity of important enzymes6
such as kinases or proteases from complex biological
mixtures. Recently, peptide nucleic acid (PNA) tags have
been used to label proteins7 and encode small molecules8
such that hybridization to an oligonucleotide microarray
(Fig. 1) localizes the tagged entity to its preprogrammed
location. An added advantage of using this strategy in
combinatorial synthesis is that libraries synthesized in a split
and mix format9 can be decoded in a single operation with
The library reported herein was targeted towards cysteine
proteases for their well know involvement in a number of
diseases10 including tumor growth,11 osteoporosis,12 inflam-
mation, neurodegenerative diseases13 apoptosis misregula-
tion,14 and infectious diseases such malaria,15 African
sleeping sickness and Chaga’s disease. It is important to
note that in at least eight cases tested, the PNA tag and its
linker did not interfere with the biological activity of the
inhibitor.
2. Results and discussion
2.1. Synthetic strategy
The general structure of the PNA-encoded library 1 is
q Supplementary data associated with this article can be found in the online
version, at doi: 10.1016/j.tet.2004.05.107
Keywords: Microarrays; Combinatorial chemistry; Protease inhibitors;
Peptide nucleic acid.
*
Corresponding author. Tel.: þ33-3-90-24-51-13; fax: þ33-3-90-24-51-
Figure 1. Hybridization of PNA-encoded library to an oligonucleotide
microarray.
12; e-mail address: winssinger@isis-ulp.org
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.05.107

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F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
Figure 2. Design and retrosynthetic analysis of PNA-encoded library.
shown in Figure 2. The inhibitor is based on an acrylate
functionality16 that upon binding in the enzymatic pocket
can be subject to a Michael addition of cysteine’s
nucleophilic thiol. The use of amino acids as the source of
diversity was designed to maximize the potential of finding
2.2. Alloc-protected PNA and Fmoc-protected inhibitor
oligomer (PG15Alloc, PG25Fmoc)
The required Fmoc protected acrylic amino acids 10 were
prepared from commercially available Fmoc-protected
amino acids 7 as shown in Scheme 1. Thus, esterification
of the acids 7 (Nle, Asp, Gln, Lys) with ethane thiol afforded
the corresponding thioesters which were reduced to
aldehydes 8 with palladium on charcoal and triethyl silane
in good yield.17 The aldehydes 8 were then condensed with
commercially available ylide 9 to afford the allyl acrylic
esters which were deprotected with palladium-catalyzed
allyl transfer to yield the desired Fmoc protected acrylic
amino acids 10. The required Alloc-protected PNA mono-
mers 14 were most conveniently obtained by exchanging the
Fmoc protecting group from commercially available PNA
monomers 11 while temporarily loading them on a 2-
chlorotrityl resin (Scheme 2) thereby avoiding all purifi-
cations and workups. Thus, acids 11 were coupled to 2-
chlorotrityl resin to obtain polymer-bound esters 12. Fmoc
deprotection with DBU18 and reprotection with allyl
chloroformate yielded the Alloc protected esters 13 which
were released from the resin using acetic acid in
trifluoroethanol/dichloromethane19 to obtained suitably
˚
a protease–ligand interaction. A spacer (ca. 20 A) contain-
ing a bis-ethyleneglycol moiety and a free amino group was
included between the inhibitor and the PNA to insure good
water solubility and reduce the risk of aggregation of the
final library. Fluorescein was selected as the label based on
the fact that it is excited with a green argon laser available in
a number of microarray scanners, it can be potentially
amplified with anti-fluorescein antibodies and it can be
easily introduced from the commercially available isothio-
cyanate (FITC). Library 1 was anticipated to come from
polymer-bound library 2 using a Rink acid labile linker, acid
labile protecting groups for the PNAs’ nucleobases as well
as for the amino acid side chains. Two orthogonal protecting
groups (PG1 and PG2) are required to carry out the
alternative oligomerization of the PNA and inhibitor from
3 using the suitably protected monomers 4–6. For this
purpose, Fmoc and Alloc protecting group were selected for
their mutual orthogonality and the accessibility of the
corresponding monomers 4–6.

F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
8679
Scheme 1. Synthesis of Fmoc protected acrylic amino acids 10. Reagents and conditions: (a) 7 (1.0 equiv.), DIC (1.6 equiv.), PhSH (1.1 equiv.), 4-DMP
(0.05 equiv.), CH2Cl2, 23 8C, 30 min, 94% for Asp, 88% for nle, 95% for Gln, 94% for Lys; (b) Et3SiH (4.0 equiv.), 5% Pd/C (0.02 equiv.), CH2Cl2, 23 8C,
90 min, 77% for 8-Nle, 87% for 8-Asp, 94% for 8-Gln, 80% for 8-Lys; (c) 9 (1.6 equiv.), EtiPr2N (1.4 equiv.), toluene, 80 8C, 90 min, 86% for Nle, 83% for
Asp, 90% for Gln, 834% for Lys; (d) Bu3SnH (3.0 equiv.), Pd(PPh3)4 (0.1 equiv.), CH2Cl2, 23 8C, 90 min, 86% for 10-Nle, 82% for 10-Asp, 89% for 10-Gln,
91% for 10-Lys.
protected PNA monomer 14. TFA cleavages with as little as
1% TFA in CH2Cl2 lead to partial Bhoc deprotection in 5 min.
With the required building blocks at hand we turned our
attention to the co-synthesis of the PNA and inhibitor. As
shown in Scheme 3, Rink resin loaded with the orthogonally
protected N-a-Fmoc-N-1-Alloc-lysine (15) was substitute
with the acrylic amino acid 10-Gln to obtain resin 16. The
Alloc group was removed and a representative codon was
introduced by reiteratively coupling PNA monomer 14-T to
obtain compound 17. Sequential Fmoc deprotection, intro-
duction of a second amino acid, Alloc deprotection and
introduction of a second codon afforded compound 18 which
was engaged in a third cycle. During this synthesis, two side
reactions that could potentially compromise the purity of a
final library were observed. First, during certain Alloc
deprotection [(Pd(PPh3)4, nBu3SnH], partial reduction of the
acrylate olefin wasobservedand second, basicconditionsused
in the Fmoc deprotection (piperidine or DBU) for compound
18 lead to partial piperazinone formation via intermediate 19.
With these results, we reasoned that it would be prudent to
reverse the protecting group strategy since the Alloc
deprotections may be carried out under acidic condition and
would avoid the piperazinone formation and, the reduced
number of Alloc deprotections should minimize the level of
acrylate reduction.
2.3. Fmoc-protected PNA and Alloc-protected inhibitor
oligomer (PG15Fmoc, PG25Alloc)
Rink resin loaded with the orthogonally protected
Scheme 2. Preparation of Alloc-protected PNA monomers 14. Reagents and conditions: (a) 11 (1.0 equiv.), EtiPr2N (1.0 equiv.), resin (2.0 equiv.), CH2Cl2,
23 8C, 3 h; (b) DBU (1.0 equiv.) CH2Cl2, 23 8C, 5 min; (c) Alloc-Cl (3.0 equiv.), EtiPr2N (4.0 equiv.), CH2Cl2, 0 8C, 2£10 min; (d) AcOH–CF3CH2OH–
CH2Cl2 (1:1:8), 60 min, precipitation in Et2O, 60% for 14-T, 46% for 14-C, 61% for 14-G, 58% for 14-A, average purity .95%.

Scheme 3. Optimization of conditions for the co-synthesis of PNA and peptides using Alloc-protected PNA and Fmoc-protected amino acids. Reagents and conditions: (a) 20% piperidine, DMF, 23 8C, 2.5 min; (b) 10-
Gln (2.0 equiv.), DIC (4.0 equiv.), HOBt (4.0 equiv.), DMF, 23 8C, 3 h; (c) nBu3SnH (10 equiv.), Pd(PPh3)4 (0.2 equiv.), CH2Cl2, 23 8C, 30 min; (d) 14-T (4.0 equiv.), EtiPr2N (4.0 equiv.), HATU (3.5 equiv.), DMF,
23 8C, 1 h.

F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
8681
Scheme 4. Preparation of Alloc-protected acrylamide 23. Reagents and conditions: (a) 20% piperidine, DMF, 23 8C, 2.5 min; H·TFA/Alloc (2.0 equiv.), DIC
(4.0 equiv.), HOBt (4.0 equiv.), DMF, 23 8C, 6 h; (c) 10-Gln (2.0 equiv.), DIC (4.0 equiv.), HOBt (4.0 equiv.), DMF, 23 8C, 6 h; (d) 20% piperidine, DMF,
23 8C, 2.5 min; Alloc-Cl (4.0 equiv.), Et/Pr2N (4.0 equiv.), DMF, 0 8C, 30 min; (d) 1% TFA, 5% Et3SiH, CH2Cl2, 23 8C, 1 h.
N-a-Fmoc-N-1-4-methyltrityl-lysine (20, Scheme 4) was
substitute with the ethyleneglycol spacer followed by
acrylic amino acid 10-Gln to obtain resin 21. The Fmoc
group was exchanged for an Alloc and the methyltrityl was
removed to obtain compound 23 as a TFA salt via 22. The
required Alloc-protected amino acids 25 with the appro-
priate side chain protecting groups could be readily obtained
from the corresponding Fmoc protected amino acids 24
(Scheme 5) by temporarily loading them on a chlorotrityl
resin. As for the PNA, this procedure avoided all work ups
and purifications while delivering the amino acids with the
appropriate side chain protecting groups. We then preceded
to the synthesis of a set of representative PNA encoded
inhibitors of which an example is Scheme 6. Thus,
alternative addition of a PNA codon by reiterative Fmoc
deprotection/coupling followed by Alloc deprotection and
amino acid coupling led after labeling with FITC and
cleavage from the resin to compound 26. The use of triethyl
silane20 rather than tributyl tin hydride for the Alloc
deprotection, while slower, proved to be more reliable and
avoided all acrylate reduction problems. The final com-
pound 26 was thus obtained from intermediate 20 in 40
steps. MALDI analysis of the final product shows the
desired compound as a single major compound (Fig. 3). The
relatively small number of truncated sequences reflects
the efficiency of the well-established coupling procedures21
and of the protecting group strategy.
2.4. Split and mix synthesis of the library
The codon system shown in Figure 4 was designed based on
the following criteria: At least 2 base pair mismatches
between each codon, no more than 6 consecutive purines
and homogeneous hybridization properties with 4-letter
codons at the extremities. The synthesis of the library was
executed as shown in Scheme 7. The completion of each
reaction was verified by mass spectroscopy analysis, a
spectra of a representative pool is shown in Figure 5
(remaining spectra’s are shown in the Supplementary
Material). Thus, the four library pools (23) were encoded
with their respective PNA sequences to obtain four pools of
27 which were mixed and subjected to a common Alloc
deprotection before being split into ten new pools. To each
Scheme 5. Exchange of protection groups for amino acid monomers.
Reagents and conditions: (a) 24 (1.4 equiv.), EtiPr2N (2.0 equiv.), resin
(1.0 equiv.) CH2Cl2, 23 8C, 3 h; (b) 20% piperidine in DMF, 23 8C, 10 min;
(c) Alloc-Cl (4.0 equiv.), EtiPr2N (4.0 equiv.), CH2Cl2, 0 8C, 30 min;
(d) 2.5% TFA in CH2Cl2, 23 8C, 30 min, 78% average yield, .95% purity
by NMR.
Figure 3. MALDI analysis of compound 26.

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F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
Scheme 6. Optimized synthesis of PNA-encode inhibitor 26. (a) 20% piperidine, DMF, 23 8C, 2.5 min; (b) 14, EtiPr2N (4.0 equiv.), 2,6-lut. (6.0 equiv.),
HATU (3.5 equiv.), DMF, 23 8C, 1 h; (c) Et3SiH (10 equiv.), Pd(PPh3)4 (0.2 equiv.), AcOH (10 equiv.), DMF/CH2Cl2 (1/1), 23 8C, 30 min; 25 (4.0 equiv.),
DIC (4.0 equiv.), HOBt (4.0 equiv.), DMF , 23 8C, 2 h; (d) 20% piperidine, DMF, 23 8C, 2.5 min; FITC (10 equiv.), lut. (10 equiv.), DMF, 23 8C 5 h; (e) TFA:
mcresol (4:1), 23 8C, 2 h, Et2O precipitation, centrifugation.
pool was added the second element of diversity followed by
its respective PNA codon to obtain ten pools of 28 each
containing 4 compounds. The pools were mixed deprotected
and re-split into 10 new pools which were subjected to
another round of diversification/encoding to yield ten pools
of 29 each containing 40 compounds. While the identity of
every component of a pool could no longer be identified by
mass spectroscopy, the absence of lower molecular weight
peaks suggests that all the steps thus far were very high
yielding as was observed in a number of test cases such as
compound 26. The ten pools of 29 were once more mixed,
deprotected, split and the last amino acid followed by the
last codon was introduced to obtain 10 pools of 30 which
were combined. MALDI analysis was no longer useful to
evaluate this last step as there are too many compounds in
the mixture. The last Fmoc was removed and the library was
labeled with fluorescein isothiocyanate (FITC). Finally, the
library was cleaved as a mixture of 4000 compounds with
TFA/cresol and precipitated in diethyl ether to obtain the
library free of protecting group byproducts. Quantification
of the fluorescein indicates a 32% recovery based on the
loading of starting resin 20.
A PNA-encoded library of 4000 compounds by split and
mix synthesis was achieved. The use of this library to profile
protease activity and identify inhibitors for subsequent
biological chemistry study will be reported shortly. While
the Alloc-protected PNA monomers proved to be less
suitable than the corresponding Fmoc monomers for the
present library, their chemistry should be applicable to other
libraries and, by virtue of their stability to basic condition,
further extend the scope of reactions to bring about
molecular diversity with PNA encoding.
3. Experimental
3.1. General techniques
All reactions were carried out under a nitrogen atmosphere
with dry, freshly distilled solvents under anhydrous
Figure 4. Sequence of the assigned codons.

F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
8683
Scheme 7. PNA-encode split and mix synthesis of library 31. Reagents and conditions: (a) 20% piperidine, DMF, 23 8C, 2.5 min; 14 (4.0 equiv.), EtiPr2N
(4.0 equiv.), HATU (3.5 equiv.), 2,6-lut. (6.0 equiv.), DMF, 23 8C, 1 h; Ac2O (4.0 equiv.), lut. (4.0 equiv.), DMF, 23 8C, 5 min; (b) Et3SiH (10 equiv.),
Pd(PPh3)4 (0.2 equiv.), AcOH (10 equiv.), DMF/CH2Cl2 (1/1), 23 8C, 30 min; (c) 25 (4.0 equiv.), DIC (4.0 equiv.), HOBt (4.0 equiv.), DMF, 23 8C, 2 h; Ac2O
(4.0 equiv.), lut. (4.0 equiv.), DMF, 23 8C, 5 min; (d) 20% piperidine, DMF, 23 8C, 2.5 min; FITC (10 equiv.), lut. (10 equiv.), DMF, 23 8C, 5 h; (e) TFA–
cresol (4:1), 23 8C, 3 h; precipitated in Et2O.

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F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
Figure 5. MALDI analysis of representative pools along the library synthesis.
conditions, unless otherwise noted. All solid phase reactions
were carried out at ambient temperature unless specified
otherwise. Tetrahydrofuran (THF), toluene and diethyl ether
(Et2O) were distilled from sodium–benzophenone, and
methylene chloride (CH2Cl2) from calcium hydride.
Anhydrous solvents were also obtained by passing them
through commercially available activated alumina columns
(Solv-Tek, Inc., VA). Yields refer to chromatographically
and spectroscopically (1H NMR) homogeneous materials,
unless otherwise stated. Substituted polystyrene resins
(100–200 mesh, 1% DVB) and amino acids were purchased
from Advanced Chemtech or Novabiochem. PNA mono-
mers and polyethyleneglycol spacer were purchased from
Applied Biosystems. Solution reactions were monitored by
thin layer chromatography (TLC) carried out on 0.25 mm
E. Merck silica gel plates (60F-254) using UV light for
visualization agent and 10% ethanolic phosphomolybdic
acid, ninhydrin or vanillin solution and heat, as developing
agents. E. Merck silica gel (60, particle size 0.040–
0.063 mm) was used for flash column chromatography.
NMR spectra were recorded on Bruker Advance-400
instruments and calibrated using residual undeuterated
solvent as an internal reference. Multiplicities were
abbreviated as: s¼singlet, d¼doublet, t¼triplet, q¼quartet,
qt¼quintet, m¼multiplet, b¼broad. IR spectra were
recorded on a Perkin–Elmer 1600 series FT-IR spectro-
meter. HRMS were recorded on a Bruker micro-TOF
instrument (ESI) while MALDIs were performed on an
Applied Biosystem Voyager.
3.2.1. Ethyl N-Fmoc-O-tButyl-thioaspartate. 94% yield
as viscous oil; Rf¼0.75 (SiO2, EtOAc–hexanes 1:1); IR
1
(KBr): 3339.9, 2976.3, 1731.1, 1503.4, 1450.0 cm21; H
NMR (400 MHz, CDCl3) d: 7.78 (2H, d, J¼7.0 Hz, ArH),
7.68 (2H, m, ArH), 7.45 (2H, m, ArH), 7.34 (2H, m, ArH),
6.15 (1H, m, NH), 4.73 (1H, m, CH), 4.60 (1H, m, Fmoc–
CH2), 4.39 (1H, m, Fmoc–CH2), 4.30 (1H, t, J¼7.0 Hz,
Fmoc–CH), 3.05 (1H, dd, J¼17.0, 4.5 Hz, CH2CO), 2.92
(2H, q, J¼7.5 Hz, SCH2CH3), 2.75 (1H, dd, J¼17.0, 4.5 Hz,
CH2CO), 1.49 (9H, s, tBu), 1.29 (3H, t, J¼7.0 Hz,
SCH2CH3); 13C NMR (100 MHz, CDCl3) d: 200.3, 170.1,
155.94, 143.9, 143.7, 141.3, 127.8, 127.1, 125.2, 125.1,
120.0, 82.0, 67.4, 57.4, 47.2, 37.5, 28.0, 23.6, 23.6, 14.4;
HRMS (ESI) C25H29NNaO5S calcd for (MNa)þ: 478.1661,
found: 478.1636.
3.2.2. Ethyl N-Fmoc-thionorleucinate. 88% yield as white
solid; Rf¼0.78 (SiO2, EtOAc–hexanes 1:1); IR (KBr):
3305.5, 2951.9, 1694.1, 1535.6, 1450.4 cm21 1H NMR
;
(400 MHz, CDCl3) d: 7.80 (2H, m, ArH), 7.52 (2H, m,
ArH), 7.44 (2H, m, ArH), 7.35 (2H, m, ArH), 5.26 (1H, d,
J¼9.0 Hz, NH), 4.53 (1H, m, CH), 4.42 (2H, m, Fmoc–
CH2), 4.28 (1H, t, J¼7.0 Hz, Fmoc–CH), 2.92 (2H, q,
J¼8.0 Hz, SCH2CH3), 1.92 (1H, m, CH2CH2CH2CH3), 1.65
(1H, m, CH2CH2CH2CH3), 1.37 (4H, m, CH2CH2CH2CH3),
1.29 (3H, t, J¼8.0 Hz, SCH2CH3), 0.94 (3H, m, CH2CH2-
CH2CH3); 13C NMR (100 MHz, CDCl3) d: 201.0, 155.8,
143.9, 143.7, 141.3, 127.7, 127.1, 125.1, 125.0, 120.0, 67.0,
61.0, 47.2, 32.6, 27.3, 23.28, 22.2, 14.5, 13.8; HRMS (ESI)
calcd for C23H27NNaO3S (MNa)þ: 420.1606, found:
420.1607.
3.2. Fmoc-protected acrylic amino acids 10
General procedure for the thioesterification. To a stirring
solution of Fmoc protected amino acid 7 in CH2Cl2 (0.2–
0.3 M) was added thioethanol (1.2 equiv.), followed by
N,N-diisopropylcarbodiimide (1.6 equiv.) and 4-DMAP
(0.05 equiv.). The reaction mixture was stirring for 25–
30 min at 23 8C then loaded directly onto a silica gel column
and eluted with EtOAc–hexanes.
3.2.3. Ethyl N-Fmoc-N-g-trityl-thioglutaminate. 95%
yield as white solid; Rf¼0.55 (SiO2, EtOAc–hexanes 1:1);
IR (KBr): 3310.5, 3056.5, 2928.3, 1679.1, 1492.4,
1
1447.2 cm21; H NMR (400 MHz, CDCl3) d: 7.79 (2H, d,
J¼7.5 Hz, ArH), 7.64 (2H, m, ArH), 7.42 (2H, m, ArH),
7.32 (2H, m, ArH), 6.85 (1H, s, NH), 7.25 (2H, m, Ar–H),
5.88 (1H, d, J¼7.0 Hz, NH), 4.56 (1H, m, CH), 4.37 (2H, m,

F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
8685
Fmoc–CH2), 4.27 (1H, t, J¼7.0 Hz, Fmoc–CH), 2.91 (2H,
q, J¼7.0 Hz, SCH2CH3), 2.42 (2H, m, CH2CH2CO), 2.23
(1H, m, CH2CH2CO), 2.04 (1H, m, CH2CH2CO), 1.27 (3H,
t, J¼7.0 Hz, SCH2CH3); 13C NMR (100 MHz, CDCl3) d:
201.0, 171.0, 156.2, 144.5, 143.9, 143.7, 141.3, 128.7,
128.0, 127.8, 127.7, 125.3, 125.2, 120.0, 70.7, 67.1, 47.3,
33.3, 27.9, 23.3, 14.4; HRMS (ESI) calcd for C41H38N2
NaO4S (MNa)þ: 677.2448, found: 677.2483.
white solid; Rf¼0.35 (SiO2, EtOAc–hexanes 1:1); IR
(KBr): 3316.9, 3057.4, 1714.0, 1671.8, 1492.5,
1
1447.6 cm21; H NMR (400 MHz, CDCl3) d: 9.47 (1H, s,
HCO), 7.70 (2H, m, ArH), 7.62 (2H, m, ArH), 7.40 (2H, m,
ArH), 7.30 (15H, m, ArH), 7.23 (2H, m, ArH), 6.83 (1H, s,
NH), 5.61 (1H, d, J¼6.0 Hz, NH), 4.49 (1H, m, CH), 4.16
(2H, q, J¼7.0 Hz, Fmoc–CH2), 4.24 (1H, t, J¼7.0 Hz,
Fmoc–CH), 2.35 (2H, m, CH2CH2CO), 2.26 (1H, m,
CH2CH2CO), 1.86 (1H, m, CH2CH2CO); 13C NMR
(100 MHz, CDCl3) d: 198.6, 170.9, 156.4, 144.5, 143.7,
141.4, 128.6, 128.0, 127.7, 127.1, 125.0, 120.0, 70.7, 66.7,
59.4, 47.3, 32.5, 24.6; HRMS (ESI) calcd for
C39H34N2NaO4 (MNa)þ: 617.2411, found: 617.2428.
3.2.4. Ethyl N-Fmoc-N-1-Boc-thiolysinate. 94% yield as
white solid; Rf¼0.51 (SiO2, EtOAc–hexanes 1:1); IR
1
(KBr): 3352.2, 2930.8, 1688.3, 1526.2, 1449.1 cm21; H
NMR (400 MHz, CDCl3) d: 7.79 (2H, d, J¼8.0 Hz, ArH),
7.66 (2H, m, ArH), 7.43 (2H, m, ArH), 7.34 (2H, m, ArH),
5.52 (1H, m, NH), 4.62 (1H, s, NH), 4.55 (1H, m, CH), 4.41
(2H, m, Fmoc–CH2), 4.27 (1H, m, Fmoc–CH), 3.14 (2H,
m, CH2CH2CH2CH2NH), 2.92 (2H, q, J¼7.5 Hz, SCH2CH3),
1.92 (1H, m, CH2CH2CH2CH2NH), 1.70 (1H, m, CH2CH2-
CH2CH2NH), 1.47 (13H, m, tBu and CH2CH2CH2CH2NH),
1.28 (3H, t, J¼7.5 Hz, SCH2CH3); 13C NMR (100 MHz,
CDCl3) d: 201.00, 156.2, 156.0, 143.9, 143.7, 141.3, 127.7,
127.1, 125.1, 120.0, 79.22, 67.1, 60.8, 47.2, 39.8, 32.2, 29.7,
28.4, 23.3, 22.3, 14.5; HRMS (ESI) C23H27NNaO3S calcd
for (MNa)þ: 535.2240, found: 535.2222.
N-a-Fmoc-N-1-Boc-lysinal (8-Lys). 80% yield as white
solid; Rf¼0.40 (SiO2, EtOAc–hexanes 1:1); IR (KBr):
3352.3, 2929.8, 1686.2, 1525.0, 1449.9 cm21 1H NMR
;
(400 MHz, CDCl3) d: 9.60 (1H, s, HCO), 7.79 (2H, d,
J¼7.5 Hz, ArH), 7.64 (2H, d, J¼7.5 Hz, ArH), 7.44 (2H,
m, ArH), 7.35 (2H, m, ArH), 5.54 (1H, s, NH), 4.62
(1H, s, NH), 4.46 (2H, m, Fmoc–CH2), 4.31 (1H, m,
CH), 4.25 (1H, t, J¼7.0 Hz, Fmoc–CH), 3.15 (2H, m,
CH2CH2CH2CH2NH), 1.96 (1H, m, CH2CH2CH2CH2NH),
1.70 (1H, m, CH2CH2CH2CH2NH), 1.46 (13H, m, tBu and
CH2CH2CH2CH2NH); 13C NMR (100 MHz, CDCl3) d:
199.4, 156.2, 143.7, 141.3, 127.1, 125.0, 120.0, 79.3, 67.0,
60.0, 47.2, 40.0, 29.8, 28.5, 28.4, 22.1; HRMS (ESI)
C26H32N2NaO5 calcd for (MNa)þ: 475.2204, found:
475.2210.
3.2.5. Aldehyde 8; general procedure for reduction of
thioester. To a stirring solution of the thioester (vide supra)
(0.2–0.3 M) and of Pd/C 5% (2 mol%) in CH2Cl2 was
added Et3SiH (4.0 equiv.). The reaction mixture was stirred
at 23 8C until TLC analysis revealed complete consumption
of the starting material (30–40 min). The crude reaction
mixture was loaded directly onto a silica gel column and
eluted with EtOAc–hexanes.
3.2.6. Allyl acrylic ester derivatives; general procedure
for the Wittig olefination. To a stirring solution of the
aldehyde 8 (0.1 M) and allylphosphonium iodide (1.6 equiv.)
in toluene at 80 8C was added EtiPr2N (1.4 equiv.). The reac-
tion mixture was stirred at 80 8C until TLC analysis revealed
complete consumption of the starting material (90–120 min).
The reaction mixture was diluted in EtOAc and washed with
0.1 N HCl, sat. NaCO3, brine, dried over MgSO4 and
purified by silica gel chromatography (EtOAc–hexanes).
N-Fmoc-O-tButyl-aspartal (8-Asp). 87% yield as viscous
oil; Rf¼0.56 (SiO2, EtOAc–hexanes 1:1); IR (KBr): 3355.9,
2977.8, 1724.4, 1515.7, 1450.0 cm21; 1H NMR (400 MHz,
CDCl3) d: 9.67 (1H, s, HCO), 7.68 (2H, d, J¼7.5 Hz, ArH),
7.63 (2H, d, J¼7.5 Hz, ArH), 7.44 (2H, m, ArH), 7.35 (2H,
m, ArH), 5.99 (1H, d, J¼7.0 Hz, NH), 4.46 (3H, m, Fmoc–
CH2 and CH), 4.30 (1H, t, J¼7.0 Hz, Fmoc–CH), 2.97 (1H,
dd, J¼17.0, 4.5 Hz, CH2CO), 2.83 (1H, dd, J¼17.0, 4.5 Hz,
CH2CO), 1.49 (9H, s, tBu); 13C NMR (100 MHz, CDCl3) d:
198.8, 170.2, 156.2, 143.8, 143.7, 141.3, 127.8, 127.1,
125.1, 120.0, 82.2, 67.3, 56.6, 47.1, 35.6, 28.0; HRMS (ESI)
C23H25NNaO5 calcd for (MNa)þ: 418.1625, found:
418.1576.
Allyl N-Fmoc-O-tButyl-acrylicaspartate. 83% yield as
viscous oil; Rf¼0.86 (SiO2, CHCl3 –MeOH 9:1); IR
(KBr): 3336.7, 2978.5, 1725.1, 1526.0 cm21 1H NMR
;
(400 MHz, CDCl3) d: 7.79 (2H, d, J¼7.5 Hz, ArH), 7.62
(2H, d, J¼7.5 Hz, ArH), 7.43 (2H, m, ArH), 7.34 (2H, m,
ArH), 6.97 (1H, dd, J¼16.0, 5.0 Hz, CHvCHCO), 5.98
(2H, m, CH2vCHCH2O and CHvCHCO), 5.70 (1H, d, J¼
8.5 Hz, NH), 5.38 (1H, dd, J¼17.0, 1.5 Hz, CH2vCHCH2-
O), 5.28 (1H, dd, J¼10.5, 1.5 Hz, CH2vCHCH2O), 4.75
(1H, m, CH), 4.68 (2H, d, J¼6.0 Hz, CH2vCHCH2O), 4.45
(2H, m, Fmoc–CH2), 4.25 (1H, t, J¼7.0 Hz, Fmoc–CH),
2.68 (1H, dd, J¼16.0, 5.0 Hz, CH2CO), 2.59 (1H, dd,
J¼16.0, 5.0 Hz, CH2CO), 1.47 (9H, s, tBu); 13C NMR
(100 MHz, CDCl3) d: 169.8, 165.5, 155.5, 146.6, 143.8,
141.3, 129.4, 128.0, 127.5, 125.6, 120.5, 118.4, 82.0, 67.0,
65.2, 48.8, 47.2, 39.4, 28.0; HRMS (ESI) C28H31NNaO6
calcd for (MNa)þ: 500.2044, found: 500.2040.
N-Fmoc-norleucinal (8-Nle). 77% yield as white solid;
Rf¼0.73 (SiO2, EtOAc–hexanes 1:1); IR (KBr): 3321.7,
2955.0, 1738.3, 1689.0, 1537.0, 1448.9 cm21 1H NMR
;
(400 MHz, CDCl3) d: 9.62 (1H, s, HCO), 7.80 (2H, m,
ArH), 7.64 (2H, m, ArH), 7.44 (2H, m, ArH), 7.35 (2H, m,
ArH), 5.34 (1H, d, J¼9.0 Hz, NH), 4.47 (2H, d, J¼7.0 Hz,
Fmoc–CH2), 4.35 (1H, m, CH), 4.26 (1H, t, J¼7.0 Hz,
Fmoc–CH), 1.95 (1H, m, CH2CH2CH2CH3), 1.66 (1H, m,
CH2CH2CH2CH3), 1.38 (4H, m, CH2CH2CH2CH3), 0.95
(3H, m, CH2CH2CH2CH3); 13C NMR (100 MHz, CDCl3) d:
199.3, 143.73, 141.3, 127.8, 127.1, 125.0, 120.0, 66.9, 60.2,
47.2, 28.9, 27.1, 22.5, 13.8; HRMS (ESI) calcd for
C21H23NNaO3 (MNa)þ: 360.1570, found: 360.1592.
Allyl N-Fmoc-acrylicnorleucinate. 86% yield as white
solid; Rf¼0.73 (SiO2, CHCl3–MeOH 9:1); IR (KBr):
1
3303.3, 2954.2, 1727.8, 1689.7, 1540.6, 1449.8 cm21; H
NMR (400 MHz, CDCl3) d: 7.80 (2H, d, J¼7.5 Hz, ArH),
N-a-Fmoc-N-g-trityl-glutaminal (8-Gln). 94% yield as
7.63 (2H, d, J¼7.5 Hz, ArH), 7.43 (2H, m, ArH), 7.35 (2H,

8686
F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
m, ArH), 6.91 (1H, dd, J¼15.5, 5.2 Hz, CHvCHCO), 5.98
(2H, m, CH2vCHCH2O and CHvCHCO), 5.38 (1H, dd,
J¼17.0, 1.5 Hz, CH2vCHCH2O), 5.29 (1H, dd, J¼10.5,
1.5 Hz, CH2vCHCH2O), 4.85 (1H, d, J¼8.0 Hz, NH), 4.68
(2H, d, J¼6.0 Hz, CH2vCHCH2O), 4.48 (2H, d, J¼7.0 Hz,
Fmoc–CH2), 4.37 (1H, m, CH), 4.25 (1H, t, J¼7.0 Hz,
Fmoc–CH), 1.63 (1H, m, CH2CH2CH2CH3), 1.55 (1H, m,
CH2CH2CH2CH3), 1.35 (4H, m, CH2CH2CH2CH3), 0.93
(3H, m, CH2CH2CH2CH3); 13C NMR (100 MHz, CDCl3) d:
165.9, 155.7, 148.6, 143.3, 141.3, 132.1, 127.7, 127.1,
125.0, 120.6, 120.0, 118.4, 66.6, 65.2, 52.0, 47.3, 34.2, 27.7,
22.3, 13.9; HRMS (ESI) calcd for C26H29NNaO4 (MNa)þ:
442.1989, found: 442.2021.
(2H, m, ArH), 7.33 (2H, m, ArH), 6.51 (1H, dd, J¼15.5,
5.5 Hz, CHvCHCO2H), 5.77 (1H, d, J¼15.5 Hz,
CHvCHCO2H), 4.48 (1H, m, CH), 4.30 (2H, m, Fmoc–
CH2), 4.23 (1H, t, J¼7.0 Hz, Fmoc–CH), 2.48 (2H, m,
CH2CO), 1.37 (9H, s, tBu); 13C NMR (100 MHz, DMSO-d6)
d: 169.8, 155.8, 144.28, 141.2, 129.4, 128.1, 127.5, 125.6,
121.8, 120.6, 120.5, 80.5, 66.0, 49.3, 47.1, 40.5, 28.1;
HRMS (ESI) C25H27NNaO6 calcd for (MNa)þ: 460.1731,
found: 460.1730.
N-Fmoc-acrylicnorleucine (10-Nle). 86% yield as white
solid; Rf¼0.43 (SiO2, CHCl3–MeOH 9:1); IR (KBr):
3401.2, 2954.8, 1701.7, 1542.6, 1409.4 cm21 1H NMR
;
(400 MHz, DMSO-d6) d: 7.89 (2H, d, J¼7.0 Hz, ArH), 7.72
(2H, d, J¼7.0 Hz, ArH), 7.51 (d, J¼8.5 Hz, 1H, NH), 7.41
(2H, m, ArH), 7.33 (2H, m, ArH), 6.53 (1H, dd, J¼15.0,
6.0 Hz, CHvCHCO2H), 5.76 (1H, d, J¼15.0 Hz, CHv
CHCO2H), 4.30 (2H, m, Fmoc–CH2), 4.23 (1H, m, Fmoc–
CH), 4.06 (1H, m, CH), 1.48 (2H, m, CH2CH2CH2CH3), 1.27
(4H, s, CH2CH2CH2CH3), 0.86 (3H, s, CH2CH2CH2CH3);
13C NMR (100 MHz, CDCl3) d: 156.1, 144.4, 144.3, 141.2,
141.1, 128.1, 127.5, 125.7, 125.6, 120.5, 65.8, 52.0, 47.2,
34.1, 28.2, 22.3, 14.5; HRMS (ESI) calcd for C23H25NNaO4
(MNa)þ: 402.1676, found: 402.1679.
Allyl N-a-Fmoc-N-g-trityl-acrylicglutamate. 90% yield as
white solid; Rf¼0.85 (SiO2, CHCl3–MeOH 9:1); IR (KBr):
3314.3, 3058.5, 2928.5, 1718.5, 1665.8, 1517.1, 1492.3,
1
1447.0 cm21; H NMR (400 MHz, CDCl3) d: 7.78 (2H, d,
J¼8.0 Hz, ArH), 7.60(2H, m, ArH), 7.41(2H, m, ArH), 7.23–
7.43 (17H, m, ArH), 6.86 (1H, dd, J¼16.0, 5.0 Hz,
CHvCHCO), 6.80(1H, s, NH), 5.96(2H, m, CH2vCHCH2O
and CHvCHCO), 5.27–5.39 (3H, m, CH2vCHCH2O and
NH), 4.67 (2H, d, J¼6.0 Hz, CH2vCHCH2O), 4.49 (1H, m,
CH), 4.41–4.54 (2H, m, Fmoc–CH2), 4.22 (1H, t,
J¼7.0 Hz, Fmoc–CH), 2.35 (2H, m, CH2CH2CO), 1.97
(1H, m, CH2CH2CO), 1.82 (1H, m, CH2CH2CO); 13C NMR
(100 MHz, CDCl3) d: 171.1, 165.7, 156.0, 147.7, 144.5,
143.3, 141.3, 132.1, 128.7, 128.0, 127.7, 127.1, 125.1,
121.1, 119.98, 118.4, 70.7, 66.6, 65.3, 51.7, 47.32, 33.4,
29.2; HRMS (ESI) calcd for C44H40N2NaO5(MNa)þ:
699.2830, found: 699.2857.
N-a-Fmoc-N-g-trityl-acrylicglutamine acid (10-Gln). 89%
yield as white-yellow solid; Rf¼0.40 (SiO2, CHCl3–MeOH
9:1); IR (KBr): 3405.8, 3057.5, 1698.8, 1494.1, 1409.2 cm21
;
1H NMR (400 MHz, DMSO-d6) d: 8.65 (1H, s, CO2H), 7.95
(2H, m, ArH), 7.73 (2H, m, ArH), 7.56 (1H, d, J¼8.5 Hz,
NH), 7.42 (2H, m, ArH), 7.34 (2H, m, ArH), 7.23 (6H, m,
ArH), 7.19 (9H, m, ArH), 6.51 (1H, dd, J¼16.0, 6.0 Hz,
CHvCHCO2H), 5.77 (1H, d, J¼16.0 Hz, CHvCHCO2H),
4.23–4.32 (3H, m, Fmoc–CH and Fmoc–CH2), 4.10 (1H,
m, CH), 2.33(2Hm, CH2CH2CO), 1.68(2H, m, CH2CH2CO);
13C NMR (100 MHz, DMSO-d6) d: 171.9, 145.4, 144.4,
144.3, 141.2, 141.1, 129.4, 129.0, 128.0, 127.9, 126.7,
125.7, 121.8, 120.6, 110.2, 69.6, 65.9, 51.8, 47.2, 33.2, 30.5;
HRMS (ESI) calcd for C41H36N2NaO5 (MNa)þ: 659.2517,
found: 659.2567.
Allyl N-a-Fmoc-N-1-Boc-acryliclysinate. 83% yield as
white solid; Rf¼0.70 (SiO2, CHCl3 –MeOH 9:1); IR
(KBr): 3347.6, 2937.0, 1716.6, 1692.8, 1528.1,
1
1450.3 cm21; H NMR (400 MHz, CDCl3) d: 7.80 (2H, d,
J¼7.0 Hz, ArH), 7.62 (2H, d, J¼7.0 Hz, ArH), 7.44 (2H, m,
ArH), 7.35 (2H, m, ArH), 5.97 (2H, m, CH2vCHCH2O and
CHvCHCO), 6.86 (1H, dd, J¼10.5, 1.5 Hz, CHvCHCO),
5.37 (1H, dd, J¼17.0, 1.5 Hz, CHvCHCO), 4.89 (1H, s,
NH), 4.67 (2H, d, J¼6.0 Hz, CH2vCHCH2O), 4.48 (2H, m,
Fmoc–CH2), 4.57 (1H, s, NH), 4.36 (1H, m, CH), 4.24 (1H,
t, J¼7.0 Hz, Fmoc–CH), 3.15 (2H, m, CH2CH2CH2CH2NH),
1.60 (2H, m, CH2CH2CH2CH2NH), 1.46 (13H, m, tBu and
CH2CH2CH2CH2NH); 13C NMR (100 MHz, CDCl3) d:
165.9, 156.2, 155.9, 148.5, 143.9, 143.8, 141.3, 132.1,
127.7, 127.1, 125.0, 124.8, 120.6, 120.0, 118.4, 79.1, 66.6,
65.2, 52.0, 47.2, 40.0, 33.8, 29.7, 28.4, 22.8; HRMS (ESI)
C31H38N2NaO6 calcd for (MNa)þ: 557.2622, found:
557.2649.
N-a-Fmoc-N-1-Boc-acryliclysine (10-Lys). 91% yield as
white solid; Rf¼0.23 (SiO2, CHCl3–MeOH 9:1); IR (KBr):
3338.4, 2934.2, 1701.5, 1526.2, 1409.8 cm21 1H NMR
;
(400 MHz, DMSO-d6) d: 7.89 (2H, d, J¼7.5 Hz, ArH), 7.72
(2H, d, J¼7.5 Hz, ArH), 7.51 (1H, d, J¼8.5 Hz, NH), 7.42
(2H, m, ArH), 7.34 (2H, m, ArH), 6.78 (1H, m, NH), 6.55
(1H, dd, J¼15.5, 6.0 Hz, CHvCHCO2H), 5.77 (1H, d,
J¼15.5 Hz, CHvCHCO2H), 4.30 (2H, m, Fmoc–CH2),
4.23 (1H, m, Fmoc–CH), 4.06 (1H, m, CH), 2.90 (2H, m,
CH2CH2CH2CH2NH), 1.48 (2H, m, CH2CH2CH2CH2NH),
3.2.7. Acrylic acid 10; general procedure for the allyl
ester deprotections. To a stirring solution of the allyl ester
(0.1 M) and Pd(PPh3)4 (0.1 equiv.) in CH2Cl2 was added
nBu3SnH (5.0 equiv.). The reaction mixture was stirred at
23 8C 90 min. The crude reaction mixture loaded directly
onto a silica gel column and eluted with MeOH–CH2Cl2.
1.37 (9H, m, tBu), 1.27 (4H, m, CH2CH2CH2CH2NH); 13
C
NMR (100 MHz, DMSO-d6) d: 156.1, 156.0, 144.34, 144.3,
141.2, 129.4, 128.1, 127.5, 125.7, 125.6, 121.8, 120.5, 77.8,
65.8, 52.1, 47.2, 40.2, 34.0, 29.7, 28.7, 23.4; HRMS (ESI)
C28H34N2NaO6 calcd for (MNa)þ: 517.2309, found:
517.2284.
N-Fmoc-O-tButyl-acrylicaspartic acid (10-Asp). 45% yield
as white-yellow solid; Rf¼0.31 (SiO2, CHCl3–MeOH 9:1);
IR (KBr): 3398.5, 2976.6, 1721.4, 1542.9, 1411.3 cm21; 1H
NMR (400 MHz, DMSO-d6) d: 7.89 (2H, d, J¼7.0 Hz,
ArH), 7.70 (2H, m, ArH), 7.62 (1H, d, J¼8.5 Hz, NH), 7.41
3.3. Alloc-protected PNA monomers 14
The reactions were carried out in parallel on an Argonaut
Quest according to the following procedure. 2-Chloro-
tritylchloride resin (75–100 mesh, 1% DVB, 1.6 mmol/g,

F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
8687
2.0 equiv.) preswelled in CH2Cl2 was treated with a solution
of acid 11 (1.0 equiv.) and EtiPr2N (1.0 equiv.) in CH2Cl2
(10 mL/g). After 3 h, the resin was filtered and washed
(CH2Cl2), and the resins were treated with DBU (1.0 equiv.)
in CH2Cl2 (10 mL/g) for 5 min to induce Fmoc deprotec-
tion. After washing (CH2Cl2), the resins were suspended in
CH2Cl2, cooled to 0 8C and EtiPr2N (4.0 equiv.) and allyl
chloroformate (3.0 equiv.) in CH2Cl2 as a 1 M CH2Cl2
solution were added. The reaction was carried out twice for
10 min at 0 8C then washed and cleaved with AcOH–
CF3CH2OH–CH2Cl2 (1:1:8) (5 mL/g of resin) for 1 h to
obtain 14 which were precipitated in diethyl ether, pelletted
by centrifugation (9000 g), resuspended in 1:1 MeCN–H2O
and lyophilized.
viscous oil; IR (KBr): 3377.8, 3246.0, 3066.7, 2944.9,
1
1731.6, 1486.3 cm21; H NMR (400 MHz, DMSO-d6) d
rotamer (8.35, 8.33, 7.83, 7.81) (1H, s, C8), 7.46 (4H, d,
J¼7.0 Hz, ArH); 7.37 (4H, t, J¼7.5 Hz, ArH), 7.31 (2H, t,
J¼7.5 Hz, ArH), (6.88, 6.87, 6.80, 6.79) (1H, s,
OCH(C6H5)2), (6.01, 5.88) (1H, m, OCH2CHvCH2),
(5.48- 5.42) (1H, m, NH), (5.36, 5.33, 5.30, 5.26) (1H, s,
NHCH2CH2N), (5.27, 5.23, 5.18, 5.16) (1H, d, J¼15.5 Hz,
CH2vCHCH2O), (5.13, 4.21) (2H, m, NCH2CON), (4.51,
4.46) (2H, m, CH2vCHCH2O), (4.84–4.45) (2H, m,
CHCH2CO), (4.30, 4.01) (2H, s, NCH2CO2H), 3.54–3.12
(4H, m, NHCH2CH2N); 13C NMR (100 MHz, DMSO-d6) d:
(171.3, 170.9), (167.5, 166.9), 156.7, 155.5, (154.2, 154.1),
149.8, 147.5, (140.9, 140.4), 134.0, (129.0, 128.9), 128.4,
126.9, (120.2, 120.0), 119.6, (117.6, 117.4), 78.5, (65.0,
64.7), 48.3, 47.3, 44.3; HRMS (ESI) calcd for C28H29N7O6:
(M2CO2þHþ): 560.2252, found: 560.2206.
3.3.1. N-(2-Allyloxyaminoethyl)-N-(thymine-1-acetyl)-
glycine (14-T). 60% yield as viscous oil; IR (KBr): 3422.0,
1
3054.7, 1707.7, 1540.9, 1474.2 cm21; H NMR (400 MHz,
DMSO-d6) d:7.32(1H, m, C6), 5.91 (1H, m, CH2vCHCH2O),
5.27 (1H, dd, J¼17.0, 1.5 Hz, CH2vCHCH2O), 5.18 (1H,
m, CH2vCHCH2O), 4.66 (1H, CH2vCHCH2O), 4.46–
4.50 (3H, m, CH2vCHCH2O and CH2CO), 4.08 (0.6H, s,
CH2CO2H), 3.98 (1.4H, s, CH2CO2H), 3.44–3.10 (4H, m,
CH2CH2), 1.17 (3H, s, Me); 13C NMR (100 MHz, DMSO-
d6) d: 170.9, 167.6, 164.8, 156.4, 151.4, 142.5, 134.1, 117.5,
108.53, 64.9, 48.0, 47.5, 47.1, 44.6, 12.3; HRMS (ESI) calcd
for C15H20NaN4O7 (MNaþ): 391.1224, found: 391.1187.
3.4. Alloc-protected amino acids 25
The reactions were carried out in parallel on an Argonaut
Quest according to the following procedure. To 2-
Chlorotritylchloride resin (75–100 mesh, 1% DVB,
1.6 mmol/g, 1.0 equiv.) swelled in CH2Cl2 (5 mL/g) was
added a solution of acid (1.4 equiv.) and EtiPr2N
(2.0 equiv.) in CH2Cl2 (5 mL/g of resin). After 3 h, the
resin was filtered and washed (CH2Cl2) and the Fmoc was
removed by standard piperidine treatment (20% in DMF,
1.5 mL, 10 min). After washing, the resins were suspended
in CH2Cl2 (5 mL/g), cooled to 0 8C and treated sequentially
with EtiPr2N (4.0 equiv.) followed by allyl chloroformate
(3.0 equiv.) as 1M solution in CH2Cl2. The reaction was
repeated once for 10 min before washing and cleaving with
TFA (2.5% in CH2Cl2, 10 mL/g) during 15 min. The
filtrates were diluted with toluene (1:1 v:v), concentrated
then lyophilised from AcCN–H2O.
3.3.2. N-(2-Allyloxyaminoethyl)-N-[4-N-(benzhydryloxy-
carbonyl)cytosine-1-acetyl]glycine (14-C). 46% yield as
viscous oil; IR (KBr): 3404.5, 3066.7. 2947.0, 1719.7,
1
1561.6, 1499.9 cm21; H NMR (400 MHz, DMSO-d6) d:
rotamer (7.90, 7.87) (d, 1H, J¼7.5 Hz, C6), 7.45 (4H, m,
ArH), 7.38 (4H, m, ArH), 7.30 (2H, m, ArH), 6.94 (1H, m,
C5), 6.81 (1H, s, CH(C6H5)2), 5.91 (1H, m, CH2vCHCH2O),
(5.27, 5.16) (2H, m, CH2vCHCH2O), (4.82, 4.62) (2H, s,
CH2CON), (4.50, 4.46) (2H, d, J¼5.5 Hz, CH2vCHCH2O),
(4.12, 3.99) (2H, s, NCH2CO2H), 3.11–3.47 (4H, m,
NHCH2CH2N); 13C NMR (100 MHz, DMSO-d6) d: (171.4,
171.0), (168.0, 167.4), 163.5, 155.4, 152.8, 151.4, 151.4,
142.3, 140.8, 134.1, 128.0, 128.3, 126.9, 117.5, (94.2, 93.1),
77.9, 64.9, 64.7, 49.9, 48.2, 47.3, 43.4; HRMS (ESI) calcd for
C28H29N5O8 (MHþ): 564.2089, found: 564.211.
3.4.1. Alloc-Ala-OH. 72% yield as viscous oil; IR (KBr):
3328.5, 2994.9, 1714.0, 1537.7, 1455.8 cm21 1H NMR
;
(400 MHz, CDCl3) d: 8.69 (1H, bs, CO2H), 6.77 (1H, s,
NH), 5.93 (1H, ddt, J¼17.0, 10.5, 5.0 Hz), 5.34 (1H, dd,
J¼17.0, 1.5 Hz, CH2vCHCH2O), 5.25 (1H, d, J¼10.5 Hz,
CH2vCHCH2O), 4.61 (1H, d, J¼5.0 Hz, CH2vCHCH2O),
4.43 (1H, m, CH), 1.49 (3H, d, J¼7.0 Hz, CH3); 13C NMR
(100 MHz, CDCl3) d: 177.5, 155.8, 132.4, 118.0, 66.5, 49.4,
18.3; HRMS (ESI) calcd for C7H11NaNO4 (MNa)þ:
196.0580, found: 196.0588.
3.3.3. N-(2-Allyloxyaminoethyl)-N-[6-N-(benzhydryloxy-
carbonyl)adenine-9-acetyl]glycine (14-A). 58% yield as
viscous oil; IR (KBr): 3419.4, 3068.7, 2935.0, 1718.6, 1522.2,
1467.1 cm21; 1H NMR (400 MHz, DMSO-d6) d: 8.60 (1H, s,
C2), 8.34 (1H, s, C8), 7.54 (4H, d, J¼7.5 Hz, ArH); 7.39
(4H, t, J¼7.5 Hz, ArH), 7.30 (2H, t, J¼7.5 Hz, ArH), 6.83
(1H, s, OCH(C6H5)2), 5.91 (1H, m, CH2vCHCH2O),
rotamer (5.36, 5.17) (s, 2H, NCH2CON), (5.32, 5.27),
(2H, m, CH2vCHCH2O), (4.53, 4.46) (2H, d, J¼5.5 Hz,
CHCH2CO), (4.33, 4.02) (2H, s, NCH2CO2H), 3.59–3.11
(4H, m, NHCH2CH2N); 13C NMR (100 MHz, DMSO-d6) d:
(171.9, 170.8), (167.5, 167.0), (156.7, 156.4), 152.7, (151.9,
151.6), 149.7, (145.7, 145.6), 141.3, 134.1, 128.9, 128.1,
126.9, 123.0, 117.5, 117.4, 77.7, (65.0, 64.7), 49.8, (48.1,
47.3), (44.5, 44.3); HRMS (ESI) calcd for C28H30N7O5:
(M2CO2þHþ): 544.2303, found: 544.2254.
3.4.2. Alloc-Arg(Pfb)-OH. 54% yield as viscous oil; IR
1
(KBr): 3341.9, 2982.9, 1670.1, 1576.1, 1457.7; H NMR
(400 MHz, CDCl3) d: 8.88 (1H, bs, CO2H), 7.60 (1H, s,
NH), 6.50 (1H, s, NH), 6.16 (1H, s, NH), 5.87 (1H, m,
CH2vCHCH2O), 5.29 (1H, d, J¼17.0 Hz, CH2vCHCH2O),
5.20 (1H, d, J¼10.0 Hz, CH2vCHCH2O), 4.54 (2H,
s, CH2vCHCH2O), 4.28 (1H, s, CH), 3.32 (2H, m,
CH2CH2CH2NH), 3.00 (2H, s, CH2C(CH3)2), 2.51 (3H, s,
ArMe), 2.47 (3H, s, ArMe), 2.12 (3H, s, ArMe), 1.91–1.73
(4H, s, CH2CH2CH2NH), 1.50 (6H, s, Me); 13C NMR
(100 MHz, CDCl3) d: 174.9, 169.2, 162.7, 156.9, 137.9,
134.7, 132.3, 129.0, 128.2, 125.3, 117.8, 87.6, 66.2,
53.2, 42.9, 41.3, 29.2, 28.5, 24.0, 19.2, 12.4; HRMS
(ESI) calcd for C23H36N4O7S (MH)þ: 511.2221, found:
511.2192.
3.3.4. N-(2-Allyloxyaminoethyl)-N-[2-N-(benzhydryloxy-
carbonyl)-guanine-9-acetyl]glycine (14-G). 61% yield as

8688
F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
3.4.3. Alloc-Asp(tBu)-OH. 84% yield as viscous oil; IR
(KBr): 3341.9, 2982.0, 1728.0, 1519.5; 1H NMR (400 MHz,
CDCl3) d: 9.87 (1H, bs, CO2H), 5.91 (1H, ddt, J¼17.0, 10.0,
5.0 Hz, CH2vCHCH2O), 5.84 (1H, d, J¼8.0 Hz, NH),
5.33 (1H, dd, J¼17.0, 1.5 Hz, CH2vCHCH2O), 5.23
(1H, dd, J¼10.0, 1.5 Hz, CH2vCHCH2O), 4.61 (3H,
m, CH2vCHCH2O and CH), 2.98 (1H, dd, J¼17.0, 5.0 Hz,
CH2OtBu), 2.79 (1H, dd, J¼17.0, 5.0 Hz, CH2OtBu), 1.45 (9H,
s, tBu); 13C NMR (100 MHz, CDCl3) d: 181.8, 176.3, 162.2,
138.5, 124.1, 88.4, 72.3, 56.5, 43.8, 34.1; HRMS (ESI) calcd
for C12H19NaNO6 (MNa)þ: 296.11045, found: 296.1119.
3.4.8. Alloc-Pro-OH. 84% yield as viscous oil; IR (KBr):
3102.6, 1704.3, 1450.4; 1H NMR (400 MHz, CDCl3) d: 9.59
(1H, bs, CO2H), 5.90 (1H, m, CH2vCHCH2O), 5.34 (2H,
m, CH2vCHCH2O), 4.65 (2H, m, CH2vCHCH2O), 4.41
(1H, m, NCHCO2H), 3.55 (2H, m, NCH2CH2CH2), 2.20
(2H, m, NCH2CH2CH2), 1.98 (2H, m, NCH2CH2CH2); 13
C
NMR (100 MHz, CDCl3) d: (155.7, 154.5), (177.9, 176.4),
132.5, (117.7, 117.3), (66.5, 66.2), (59.2, 58.6), (46.9, 46.6),
(30.9, 29.4), (24.3, 23.4); HRMS (ESI) calcd for C9H14NO4
(MH)þ: 200.0917, found: 200.0898.
3.4.9. Alloc-Ser(tBu)-OH. 70% yield as viscous oil; IR
(KBr): 3331.5, 2976.2, 1729.8, 1517.6; 1H NMR
(400 MHz, CDCl3) d: 5.93 (1H, ddt, J¼17.0, 10.5,
5.5 Hz, CH2vCHCH2O), 5.65 (1H, d, J¼8.5 Hz, NH),
5.35 (1H, d, J¼17.0 Hz, CH2vCHCH2O), 5.25 (1H, d,
J¼10.5 Hz, CH2vCHCH2O), 4.62 (2H, d, J¼5.5 Hz,
CH2vCHCH2O), 4.49 (1H, m, CH), 3.91 (1H, dd, J¼9.0,
3.5 Hz, CH2OtBu), 3.61 (1H, dd, J¼9.0, 3.5 Hz, CH2-
OtBu), 1.19 (9H, s, tBu); 13C NMR (100 MHz, CDCl3) d:
175.1, 156.1, 132.5, 118.0, 74.0, 66.0, 61.7, 54.2, 27.2;
HRMS (ESI) calcd for C11H19NaNO (MNa)þ: 268.1155,
found: 268.1135.
3.4.4. Alloc-His(Boc)-OH. 85% yield; IR (KBr): 3329.9,
2935.0, 1720.1, 1528.2; 1H NMR (400 MHz, CDCl3) d: 8.18
(1H, s, CHNCHN), 7.25 (1H, s, CHNCHN), 5.95 (1H, m,
CH2vCHCH2O), 5.86 (1H, d, J¼10.5 Hz, NH), 5.35 (1H,
d, J¼17.0 Hz, CH2vCHCH2O), 5.25 (1H, d, J¼10.5 Hz,
CH2vCHCH2O), 4.61 (2H, d, J¼5.5 Hz, CH2vCHCH2O),
4.56 (1H, m, CH), 3.14 (2H, m, CHCH2C), 1.63 (9H, s,
tBu); 13C NMR (100 MHz, CDCl3): 173.0, 162.3, 155.7,
146.2, 136.8, 132.7, 117.8, 115.5, 86.6, 65.7, 53.2, 29.7,
27.8; HRMS (ESI) calcd for C15H22N3O6 (MH)þ: 340.1503,
found: 340.1513
3.4.5. Alloc-Lys(Boc)-OH. 91% yield as viscous oil; IR
(KBr): 3346.0, 2935.0, 1701.0, 1522.2; 1H NMR (400 MHz,
CDCl3) d: 6.36 (1H, s, NH), 5.91 (1H, m, CH2vCHCH2O),
5.73 (1H, d, J¼8.0 Hz, NH), 5.31 (1H, d, J¼17.0 Hz,
CH2vCHCH2O), 5.22 (1H, d, J¼10.0 Hz, CH2vCHCH2O),
4.58 (2H, d, J¼5.0 Hz, CH2vCHCH2O), 4.37 (1H, m,
CH), 3.11 (2H, m, CH2CH2CH2CH2NH), 1.88 (1H, m,
CH2CH2CH2CH2NH), 1.76 (1H, m, CH2CH2CH2CH2NH),
1.45 (13H, s, tBu and CH2CH2CH2CH2NH); 13C NMR
(100 MHz, CDCl3) d: 175.9, 175.5, 156.2, 132.6, 117.8,
79.6, 65.9, 53.6, 40.0, 31.8, 29.5, 28.4; HRMS (ESI) calcd
for C15H26NaN2O6 (MNa)þ: 353.1683, found: 353.1705.
3.4.10. Alloc-Val-OH. 85% yield as viscous oil; IR (KBr):
3329.9, 2970.9, 1707.6, 1534.3, 1468.3 cm21 1H NMR
;
(400 MHz, CDCl3) d: 6.34 (1H, d, J¼8.5 Hz, NH), 5.95
(1H, ddt, J¼17.0, 10.5, 5.5 Hz, CH2vCHCH2O), 5.34 (1H,
dd, J¼16.0, 1.0 Hz, CH2vCHCH2O), 5.25 (1H, dd,
J¼10.5, 1.0 Hz, CH2vCHCH2O), 4.61 (2H, d, J¼5.5 Hz,
CH2vCHCH2O), 4.36 (1H, dd, J¼9.0, 4.4 Hz, CH), 2.26
(1H, m, CH(CH3)2), 1.03 (3H, d, J¼7.0 Hz, CH(CH3)2),
0.96 (3H, d, J¼7.0 Hz, CH(CH3)2); 13C NMR (100 MHz,
CDCl3) d:176.9, 156.3, 132.5, 118.0, 58.8, 66.1, 31.0,
(18.99, 17.32); HRMS (ESI) calcd for C9H15NaNO4
(MNa)þ: 202.1074, found: 202.1082.
3.4.6. Alloc-Nle-OH. 73% yield as viscous oil; IR (KBr):
3329.9, 2959.1, 1715.5, 1537.8, 1456.4 cm21 1H NMR
;
3.5. Library synthesis
(400 MHz, CDCl3) d: 9.47 (1H, bs, CO2H), 6.34 (1H,
d, J¼8.5 Hz, NH), 5.95 (1H, ddt, J¼16.5, 10.5, 5.5 Hz),
CH2vCHCH2O), 5.34 (1H, dd, J¼16.5, 1.5 Hz, CH2v
CHCH2O), 5.25 (1H, dd, J¼10.5, 1.5 Hz, CH2vCHCH2O),
4.61 (2H, d, J¼5.5 Hz, CH2vCHCH2O), 4.39 (1H, m,
CH), 1.89 (1H, m, CH2CH2CH2CH3), 1.73 (1H, m,
CH2CH2CH2CH3), 1.37 (4H, m, CH2CH2CH2CH3), 0.93
(3H, t, J¼7.0 Hz, CH2CH2CH2CH3); 13C NMR (100 MHz,
CDCl3) d: 177.5, 156.0, 132.5, 118.0, 53.7, 66.0, 32.0, 27.2,
22.2, 13.8; HRMS (ESI) calcd for C10H17NaNO4 (MNa)þ:
238.1049, found: 238.1035.
Rink amide resin (0.8 mmol/g) was loaded with a
substoichiometric amount of N-a-Fmoc-N-1-4-methyl-
trityl-lysine (0.2 mmol/g), DIC (1.0 equiv.), HOBt
(1.0 equiv.) in DMF (10 mL/g) for 12 h and acetic
anhydride (5.0 equiv.) with 2,6-lutidine (5.0 equiv.) was
added to cap the resin (2 h). Four 280 mg portions of this
resin were distributed into 5 mL tubes on an Argonaut Quest
210 and, after 30 min swelling in CH2Cl2, the resins were
deprotected with 20% piperidine in DMF (3 mL) for
2.5 min. An automated washing procedure was used after
each step involving four DMF washes (2 mL/g of resin)
with a 30 s agitation (no contracting solvents such as
methanol or diethyl ether were used). The resins were then
treated with Fmoc-8-amino-3,6-dioxaoctanoic acid
(2.0 equiv.), HOBt (4.0 equiv.) and DIC (4.0 equiv.) in
DMF (2 mL) for 6 h. Subsequent Fmoc deprotection (20%
piperidine in DMF, 3 mL) followed by coupling of each
pool to an acrylic amino acids 10 (2.0 equiv.), DIC
(4.0 equiv.), HOBt (4.0 equiv.) afforded four pools of
intermediates 21. The Fmocs were removed (20% piperi-
dine in DMF, 3 mL) and each pool, in CH2Cl2 at 0 8C, was
treated sequentially with EtiPr2N (4.0 equiv.) and Alloc-Cl
(4.0 equiv.) for 30 min. The pools were then treated with a
1% TFA solution containing 5% Et3SiH for 1 h to obtain the
3.4.7. Alloc-Phe-OH. 84% yield as viscous oil; IR (KBr):
1
3329.9, 3042.7, 2947.0, 1718.6, 1522.2, 1492.6 cm21; H
NMR (400 MHz, CDCl3) d: 7.32 (3H, m, ArH), 7.21 (2H,
m, ArH), 6.27 (1H, d, J¼8.0 Hz, NH), 5.91 (1H, ddt,
J¼17.0, 10.5, 5.5 Hz, CH2vCHCH2O), 5.26 (2H, m,
CH2vCHCH2O), 4.72 (1H, dt, J¼8.0, 6.0 Hz, CH), 4.59
(2H, d, J¼5.5 Hz, CH2vCHCH2O), 3.24 (1H, dd, J¼14.0,
6.0 Hz, CH2), 3.14 (1H, dd, J¼14.0, 6.0 Hz, CH2); 13C
NMR (100 MHz, CDCl3) d: 176.3, 155.8, 135.5, 132.4,
129.3, 128.7, 127.3, 118.0, 66.0, 54.5, 37.7; HRMS (ESI)
calcd for C13H15NaNO4 (MNa)þ: 272.0893, found:
272.0910.

F. Debaene et al. / Tetrahedron 60 (2004) 8677–8690
8689
polymer bound intermediates 23 which were encoded
References and notes
according to the general procedure below. The four pools
were then combined for the Alloc deprotection according to
the general procedure below and redistributed in ten 5 mL
tubes. All resin manipulations and distributions were carried
out as slurries using an isopycnic mixture of CH2Cl2 and
DMF and an eppendorf repeater for distribution. The ten
pools were subjected to their respective amino acid coupling
followed by the encoding according to the general
procedures to obtain ten pools of 28. Two more iteration
of this cycle led to 10 pool of intermediate 30 which were
mixed, and Fmoc deprotected (20% piperidine) followed
by treatment of fluoresein isothiocyanate (4.0 equiv.) and
2,6-lutidine in DMF (10 mL/g) for 5 h while shielding the
reaction from light with aluminum foil to obtain after
filtration and washing a distinctly yellow/orange polymer.
This resin was treated with TFA–cresol (4:1, 10 mL)
for 3 h then precipitated in Et2O (200 mL) and pelleted
by centrifugation (9800 g). The pellet was taken back
up in neat TFA (7 mL) and reprecipitated and pelleted
in Et2O (200 mL). The pellet was washed several times
with Et2O than dissolved in a 1:1 mixture of AcCN–
H2O (1:1, 20 mL) and lyophilized to obtain 390 mg of a
bright yellow powder.
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and treated with a premixed (5 min) solution of PNA
monomer 11 (4.0 equiv.), EtiPr2N (4.0 equiv.), HATU
(3.5 equiv.) and 2,6-lutidine (6.0 equiv.) for 1 h. The
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capped with acetic anhydride (5.0 equiv.) and 2,6-lutidine
(5.0 equiv.) in DMF (10 mL/g). This cycle was repeated 4
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´
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in DMF (10 mL/g of resin) was premixed for 5 min than
added to the resin and the reaction was continued for 2 hr
then capped by the addition of acetic anhydride (5.0 equiv.)
and 2,6-lutidine (5.0 equiv.).
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4. Supplementary Material
Mass spectroscopy data of compounds 27–29 as well as
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Acknowledgements
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We thank Human Frontier Science Program for their
support of this work (HFSP 0080/2003).

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