Expanding the scope of PNA-encoded synthesis (PES): Mtt-protected PNA fully orthogonal to Fmoc chemistry and a broad array of robust diversity-generating reactions

Article abstract of DOI:10.1002/chem.201201337

Nucleic acid-encoded libraries are emerging as an attractive and highly miniaturized format for the rapid identification of protein ligands. An important criterion in the synthesis of nucleic acid encoded libraries is the scope of reactions that can be used to introduce molecular diversity and devise divergent pathways for diversity-oriented synthesis (DOS). To date, the protecting group strategies that have been used in peptide nucleic acid (PNA) encoded synthesis (PES) have limited the choice of reactions used in the library synthesis to just a few prototypes. Herein, we describe the preparation of PNA monomers with a protecting group combination (Mtt/Boc) that is orthogonal to Fmoc-based synthesis and compatible with a large palette of reactions that have been productively used in DOS (palladium cross-couplings, metathesis, reductive amination, amidation, heterocycle formation, nucleophilic addition, conjugate additions, Pictet-Spengler cyclization). We incorporate γ-modifications in the PNA backbone that are known to enhance hybridization and solubility. We demonstrate the robustness of this strategy with a library synthesis that is characterized by MALDI MS analysis at every step. Keeping track of a library: A strategy that is compatible with a large palette of reactions that have been productively used in diversity-oriented synthesis (palladium cross-couplings, metathesis, π-allylation, CuAAC, reductive amination, amidation, heterocycle formation, nucleophilic addition, conjugate additions, Pictet- Spengler cyclization) is described (see figure). Copyright

Full text of DOI:10.1002/chem.201201337

FULL PAPER
DOI: 10.1002/chem.201201337
Expanding the Scope of PNA-Encoded Synthesis (PES): Mtt-Protected PNA
Fully Orthogonal to Fmoc Chemistry and a Broad Array of Robust Diversity-
Generating Reactions
Dalila Chouikhi, Mihai Ciobanu, Claudio Zambaldo, Vincent Duplan,
Sofia Barluenga, and Nicolas Winssinger*[a]
Abstract: Nucleic acid-encoded libra-
ries are emerging as an attractive and
highly miniaturized format for the
rapid identification of protein ligands.
An important criterion in the synthesis
of nucleic acid encoded libraries is the
scope of reactions that can be used to
introduce molecular diversity and
devise divergent pathways for diversi-
ty-oriented synthesis (DOS). To date,
the protecting group strategies that
have been used in peptide nucleic acid
(PNA) encoded synthesis (PES) have
limited the choice of reactions used in
the library synthesis to just a few pro-
totypes. Herein, we describe the prepa-
ration of PNA monomers with a pro-
tecting group combination (Mtt/Boc)
that is orthogonal to Fmoc-based syn-
thesis and compatible with a large pal-
cross-couplings, metathesis, reductive
amination, amidation, heterocycle for-
mation, nucleophilic addition, conju-
gate additions, Pictet–Spengler cycliza-
tion). We incorporate g-modifications
in the PNA backbone that are known
to enhance hybridization and solubility.
We demonstrate the robustness of this
strategy with a library synthesis that is
characterized by MALDI MS analysis
at every step.
AHCTUNGTRENGNeUN tte of reactions that have been pro-
ductively used in DOS (palladium
Keywords: combinatorial chemis-
try · drug discovery · multicompo-
nent reactions · protecting groups ·
synthetic methods
Introduction
successfully utilized for the encoding: DNA[3] and PNA[4]
(peptide nucleic acid[5]). Whereas the general idea of encod-
ing the synthesis of small molecule libraries with DNA was
advanced at the onset of combinatorial chemistry,[6] its prac-
tical implementation was not straightforward. Break-
throughs in the area have come from innovative strategies
to achieve chemical transformations of suitably derivatized
DNA by using templated chemistry or sorting methods[7]
(see also references cited in ref. [2]). Concurrently, PNA-en-
coded synthesis (PES) was developed[2d,8] (see also referen-
ces cited in ref. [3]) based on the premise that its peptide-
based chemistry is more robust than the phosphoramidite
chemistry of DNA and should be compatible with classical
split and mix combinatorial synthesis (Figure 1). Multiple li-
braries have now been reported with PES. The libraries can
either be used directly for affinity selection, or displayed on
DNA arrays or on DNA-templates in solution. Either
format also offers the possibility of combining libraries of
fragments.[4a,e,f] Ultimately, an important criterion for nucleic
acid encoded libraries will be the quality and the scope of
the chemistry available to access a relevant diversity space.
To date, the protecting group strategies that have been used
in PES have limited the scope of reactions used in the li-
brary synthesis to just a few prototypes. Herein, we describe
the preparation of PNA monomers with a protecting group
combination (Mtt/Boc) that is orthogonal to Fmoc-based
synthesis and is compatible with a broad array of complexi-
ty-building reactions.
The discovery of small molecules that perturb a given bio-
logical pathway by binding to a specific target is at the core
of chemical biology and drug discovery. Whereas high-
throughput screening (HTS) approaches have proven effec-
tive, there remains a need to accelerate the discovery of bio-
active compounds and reduce the overall cost.[1] Encoding
technologies based on nucleic acid tags are emerging as a
potential way to achieve this goal.[2] Nucleic acid encoding
platforms have important assets: 1) the nucleic acid tag ena-
bles decoding of selected compounds from large mixtures;
2) it offers a highly miniaturized format (screens are typical-
ly carried out with just a few micrograms of protein without
the need for complex robotics); 3) it does not require intrin-
sic knowledge of the target (as opposed to displacement
assays); 4) amplification of the tags followed by translation
into the encoded molecules enables iterative cycles of selec-
tion/amplification. Two nucleic acid platforms have been
[a] D. Chouikhi, M. Ciobanu, C. Zambaldo, V. Duplan, Dr. S. Barluenga,
Prof. N. Winssinger
Institut de Science et Ingꢀnierie Supramolꢀculaires
ISIS - UMR 7006
Universitꢀ de Strasbourg - CNRS
8 allꢀe Gaspard Monge, 67000 Strasbourg (France)
Fax : (+33)368855112
E-mail: winssinger@unistra.fr
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envisioned using a 4-methyl trityl (Mtt)[9] group, which we
anticipated to be compatible with the majority of robust re-
actions that have proven to be useful in diversity oriented
synthesis (DOS). Furthermore, the Mtt group is well-known
to be orthogonal to Fmoc and should allow the cosynthesis
with Fmoc-protected building blocks.[10] We had established
that Boc-protected nucleobases are stable to 1% trifluoro-
acetic acid (TFA)[8c] and, hence, should be compatible with
Mtt chemistry and Rink resin. Monomethoxy trityl (Mmt)
protected PNA monomers in combination with base-labile
acyl groups were previously reported to offer a synthetic
strategy similar to standard oligonucleotide synthesis condi-
tions in the preparation of PNA–DNA chimera.[11] However,
amino-Mmt groups are extremely labile and require special
precautions that are not practical for PES. The synthesis of
Mtt-protected monomers is shown in Scheme 1. Monopro-
tection of ethylene diamine with Mtt-Cl followed by alkyla-
tion with methyl bromoacetate afforded the backbone 2 in
good yield with simple scalable procedures. Acylation with a
range of Boc-protected nucleobases followed by saponifica-
tion afforded four different monomers (5A, 5C, 5G, and
5T) in good yield.
One issue with PNA-encoded libraries is the poor solubili-
ty of the PNA oligomers. This led us to explore the incorpo-
ration of modified PNA containing a lysine or arginine resi-
due in place of the glycine of the PNA backbone[8a] (modifi-
cation at the a position of the PNA backbone). Recent stud-
ies have highlighted the benefits of modifications at the g
position of the PNA backbone.[12] Such modifications confer
Figure 1. PNA-encoded synthesis (PES) by split and mix and reformat-
ting of the library into a spatially addressable microarray or combinatori-
al display of fragments.
Results and Discussion
Several strategies have been
reported for PES using com-
mercially available Fmoc-pro-
tected PNA monomers[4a,b,
d,e,8a,b]
(Bhoc protected nucleo-
bases), Dde-protected mono-
mers[4h–l] (trityl protected nu-
cleobases), and Azoc-protected
monomers (Boc-protected nu-
cleobases).[8c] Nonetheless, with
the exception of one exam-
ple,[4f] these strategies have
been restrained to peptide li-
braries and the orthogonality
of the protecting group does
limit a broader scope. For ex-
ample, the sensitivity of Fmoc
to basic conditions is a severe
limitation; the sensitivity of
Dde to nucleophiles is restric-
tive, and the rapid reaction of
Azoc with phosphane pre-
cludes most palladium-cata-
lyzed reactions. To this end, we
Scheme 1. Synthesis of MttACHTGNUTERNNU(G Boc)-protected monomers 5 (A, C, G, T) and 10 (A, C, G, T). Reagents and condi-
tions: a) MttCl (1.0 equiv), Py, CH2Cl2, 4 h, 97%; b) Et3N (1.0 equiv), BrCH2CO2Me (1.1 equiv), CH2Cl2, 3 h,
70%; c) 3T, 3C, 3A, or 3G (1.5 equiv), DIPEA (1.8 equiv), 2,6-lutidine (1.5 equiv), HATU (1.4 equiv), DMF,
12 h, 83% for 4C, 95% for 4T, 88% for 4G and 85% for 4A; d) LiOH (4.0 equiv), dioxane/H2O (3:1), 2 h,
82% for 5C, 83% for 5T, 75% for 5G and 78% for 5A; e) NMM (1.0 equiv), C4H9COCl (1.0 equiv), DME,
then NH4OH (4.0 equiv), 4 h; DBU (0.4 equiv), CH2Cl2, 6 h, 80%; f) LAH (7.0 equiv), Et2O, 48 h, 73%;
b) Et3N (1.0 equiv), BrCH2CO2Me (1.1 equiv), CH2Cl2, 3 h, 68%; c) 3T, 3C, 3A or 3G (1.5 equiv), DIPEA
(1.8 equiv), 2,6-lutidine (1.5 equiv), HATU (1.4 equiv), DMF, 12 h, 75% for 9C, 98% for 9T, 90% for 9G,
and 78% for 9A; d) LiOH (4.0 equiv), dioxane/H2O (3:1), 2 h, 82% for 10C, 80% for 10T, 86% for 10G, and
87% for 10A. DIPEA=N,N-diisopropylethylamine; DBU=1,8-diazabicyclo
methylformamide; HATU=2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate
methanaminium; Mtt=4-methyltrityl; NMM=N-methylmorpholine; Py=pyridine.
ACHTUNGTNER[NUNG 5.4.0]undec-7-ene; DMF=di-
A
ACHTUNGTRENNUNG
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Expanding the Scope of PNA-Encoded Synthesis
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a helicity to the PNA, which
further enhances its affinity
and specificity while providing
a
handle for solubilizing
groups (g-modification origi-
nating from l-amino acids pro-
mote right handedness).[12c] To
this end, we set out to synthe-
size the serine-modified PNA
monomers 10 (Scheme 1).
Starting from Fmoc-SerACTHNUTRGNE(NUG tBu)-
OH, the serine amide 7 was
obtained in excellent yield.
Amine protection followed by
reduction of the amide with
lithium aluminum hydride pro-
vided the required Mtt-pro-
tected diamine. This strategy
was selected because it is
known to be resistant to epi-
merization.[13] The remaining
alkylation, acylation, and hy-
drolysis following the same
procedures used for the un-
modified PNA monomers af-
Scheme 2. Optimization of coupling and deprotection conditions.
forded
equally
efficiently
modified monomers 10A, 10C,
10G, and 10T.
With the PNA monomers in
hand, we next turned our at-
tention to the synthesis of
oligomers and to the optimiza-
tion of the activation and de-
protection steps. Classic car-
boxylic activation with uroni-
um (HATU, HBTU, HCTU,
TNTU, TSTU), phosphonium
(BOP)
and
carbodiimide
(DIC) conditions were evalu-
ated. To assess the relative ef-
ficiency of the reaction under
conditions that could be readi-
ly adapted to automated syn-
thesis, the reaction yields were
compared by LC/MS analysis
of crude reaction mixtures by
using one equivalent each of
monomer 10A, activator, and
amine (Scheme 2). Gratifying-
ly, the potential side reaction
leading to
a six-membered
lactam was not observed under
any conditions. The best yields
were observed with the suc
ACHTUNGTNERcNUNG in-
ACHTUNGTRENNUNGimide esters (TSTU and
TNTU, 71 and 82% yield, re-
spectively). Assessing the next
Figure 2. Automated synthesis of an 18 mer PNA. Analysis of the crude cleavage product by LC/MS (top) and
MALDI MS analysis (bottom). Bold letters denote serine-modified residues.
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N. Winssinger et al.
step in the sequence, the deprotection of Mtt was complete
in under 5 min by using either 1% TFA in dichloromethane
or in 10 min with neat hexafluoroisopropanol (HFIP). How-
ever, neither conditions were deemed ideal; TFA is corro-
sive and is not recommended for use in automated synthe-
sizers, whereas HFIP does not swell well the resin. Diluting
the HFIP with dichloroethane gave comparably fast reaction
while providing a better resin swelling profile. Finally, to
keep the amine resulting from the deprotection protonated
as a salt, a small excess of 1-hydroxybenzotriazole (HOBt)
was included in the deprotection solution. This proved to be
beneficial for the Mtt deprotection as well, affording com-
plete deprotection in less than 5 min. We then used the best
conditions identified in the solid-phase synthesis of oligo-
higher specificity for the perfect match versus mismatched
compared with unmodified (average ratio of PM/MM for
modified PNA and unmodified PNA are 3.2 and 2.1, respec-
tively). These results are in good agreement with previous
reports on the benefit of including g-modifications originat-
ing from l-amino acids.
We next investigated the scope of the chemistry that
could be achieved with PES, focusing particularly on reac-
tions that have proven to be important for library synthesis
in providing diversity and complexity.[14] To this end, a resin
containing a first codon with the four different possible nu-
cleobases (TGCA)[15] including a serine modified derivative
(A) was used to evaluate different reactions (Figure 4). The
outcomes of all reactions were assessed by MALDI and LC/
MS analysis of the crude reaction cleavage product. Palladi-
um-catalyzed reactions certainly stand as one of the most
utilized reaction type in library synthesis. Resin 15 contain-
ing an Mtt-protected PNA tag was derivatized with 4-iodo-
benzoic acid to obtain 16, which was engaged in Suzuki–
Miyaura cross-coupling[16] with styrenyl boronic acid under
classical coupling conditions (palladium tetrakis) to afford a
complete cross-coupling reaction (17). Heck reaction[17] was
equally productive, affording 18 in excellent yield. Stille
cross-coupling[18] also proceeded as anticipated to yield the
expected biaryl 19. Palladium-catalyzed p-allylation[19] reac-
tions were also found to be very productive. Treatment of
PNA-tagged amine 20 with an excess of allyl acetate in the
presence of 10% palladium-Dppe catalyst cleanly afforded
the expected double allylation product 21. Reaction of a
PNA-tagged secondary amine (proline: 22) with a more con-
gested p-allyl system (1-phenyl allyl acetate) still afforded
the desired compound 23 in good yield (more than 85%
conversion, regioselectivity extrapolated from the same re-
action performed in solution). Ring-closing metathesis
(RCM) reactions have been used extensively in DOS, and
ruthenium-based catalysts are known to be compatible with
peptides.[20] Indeed, substrate 24 containing two terminal al-
kenes underwent smooth RCM with first generation Grubbs
catalyst to yield 25.[21] The Cu-catalyzed azide alkyne cyclo-
addition[22] has proven to be one of the most robust reac-
tions, and azide substrate 26 was converted into the triazole
27 in excellent yield. Beyond transition-metal-catalyzed re-
actions, the aldehyde function is a versatile starting point for
diversification. PNA-encoded aldehyde 28 could be smooth-
ACHTUNGTRENNUNGmers containing one modified PNA monomer per codon. As
shown in Figure 2, the crude cleavage of an 18-mer PNA
showed a single major peak, corresponding to the desired
oligomer, attesting to the efficiency of the chemistry.
We next investigated the impact of serine-modified PNA
in the context of microarray hybridization. To this end, mi-
croarrays containing combinatorial permutations of four sets
of codons representing 625 combinations of 14 mers were
used. The arrays are particularly informative with regards to
sequence selectivity because, for any given sequence on the
array, there are 19 other sequences that share three out of
the four codons. We chose a sequence that had previously
been identified as particularly promiscuous due to a long
stretch of GC nucleotides and measured the fluorescent in-
tensity for the perfect match as well as the brightest mis-
match starting at 2 nm with two-fold dilutions (i.e., 1, 0.5,
0.25 and 0.125 nm, and 62.5, 31.2, and 16 pM). As shown in
Figure 3, the modified PNA was three to five times more in-
tense at a given concentration, resulting in more sensitive
detection threshold (31.2 pM) relative to unmodified PNA
(125 pM). More importantly, the modified PNA showed a
ly engaged in reductive amination using NaBHACHTUNGTRENNUNG(OAc)3,
Horner–Wadsworth–Emmons olefination (phosphonate,
DBU, LiCl), or Knoevenagel condensation using piperidine
to obtain 29, 30 and 31, respectively, in excellent yields. In-
terestingly, performing the Knoevenagel condensation with
1,3-cyclohexanedione yielded a double addition product re-
sulting in formation of tetrahydroxantene 32. Aldehyde 28
could also be engaged in the Baylis–Hillman reaction[23]
with acrylates using quinuclidine to afford the addition
product 33 in very good yield. Substitution reactions were
also examined as an entry into privileged heterocyclic
motifs.[24] Starting with chloroacetamide resin 34, the chlor-
ide could be readily displaced by nucleophilic thiols, such as
Figure 3. Left: Plot of the fluorescent intensities measured for the perfect
matched hybridization (PM) and brightest mismatched hybridization
(MM) for serine modified PNA (black) and unmodified PNA (gray);
Right: Images of the array at 0.5 nm for the unmodified PNA (left) and
ser-modified PNA (right). Unmodified PNA sequence: K-GAA CCC
GGT GGA CG-K
ACHTUNGTRENNUNG(Cy3); K-GAA CCC GGT GGA CG-KACHTUNGTERN(NUGN Cy3). Bold
letters denote serine-modified residues.
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Expanding the Scope of PNA-Encoded Synthesis
FULL PAPER
thiophenol, to afford 36. Conversely, the chloride could be
displaced by hydrazine, which could then be further reacted
with 1,3-diketones to afford pyrazoles such as 37. Nucleo-
philic aromatic substitutions were also found to proceed
smoothly. Reaction of PNA-encoded amine 38 with 4-chlo-
ACTHNGUTRENNUG ACHTUNGRENqNUG uinazoline using DIPEA as a base afforded the desired
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
yield. Finally, we investigated Pictet–Spengler cyclizations,[25]
which are among the most prominent synthetic approaches
to polyheterocyclic compounds. Whereas this reaction is per-
formed under acidic conditions and would thus not be com-
patible with Mtt or the Rink linker, we reasoned that it
could be achieved concomitantly with the final cleavage.
Thus, taking 3,4-dimethoxyphenyl alanine and coupling di-
methoxy valerate afforded resin 42. Upon treatment with
TFA to promote the cleavage and global deprotection, the
cyclization did take place as anticipated, affording 43. Alter-
natively, a suitably substituted amine on the resin (such as
tryptophan) could be condensed with an aldehyde; the re-
sulting imine was then treated with TFA to obtain the cycli-
zation product 45 in excellent yield. Thus, Pictet–Spengler
cyclizations are possible from both imine and N-acyliminium
electrophiles in the context of PES.
We then turned our attention to the validation of this
chemistry in the context of a library synthesis. Fmoc-protect-
ed fragments certainly represent one of the most diverse
Figure 4. Diversity-building reactions in PES. Reagents and conditions:
a) 4-iodobenzoic acid (5.0 equiv), HOBt (5.0 equiv), DIC (15 equiv),
NMP, 2 h; b) [Pd
(8.0 equiv), Na2CO3 (9.0 equiv), DMF/H2O (4:1), 808C, 12 h; c) Pd-
(OAc)2 (40%), P(o-tol)3 (50%), TBACl (cat), methyl acrylate (30 equiv),
DMF/H2O/Et3N (9:1:1), 808C, 12 h; d) [Pd2A(dba)3] (20%), tri(2-furyl)-
phosphane (40%), LiCl (cat), PhSn(nBu)3 (6.0 equiv), NMP, 808C, 12 h;
(dba)3] (10%), Dppe (20%), allyl acetate (3.0 equiv), CH2Cl2, 5 h;
(dba)3] (10%), Dppe (20%), 1-phenylallyl acetate (3.0 equiv),
ACHTUNGTREN(UNNG PPh3)4] (20%), trans-2-phenylvinyl boronic acid
A
ACHTUNGTRENNUNG
C
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
e) [Pd
2ACHTUNGTRENNUNG
f) [Pd CHTUGNTRENNUNG
2A
CH2Cl2, 3 h; g) Grubbs I (50%), DCE, 508C, 12 h; h) phehylacetylene
(15 equiv), sodium ascorbate aq. (15 equiv), copper sulfate aq.
(0.50 equiv), TBTA (1.0 equiv), NMP, 16 h; i) benzyl amine (15 equiv),
NaBHACTHUNTRGNENG(U OAc)3 (30 equiv), TMOF, 12 h; j) triethyl phosphonoacetate
(4.0 equiv), DBU (5.0 equiv), LiCl (cat), DMF, 12 h; k) ethyl benzoylace-
tate (4.0 equiv), piperidine (5.0 equiv), DMF, 12 h; l) 1,3-cyclohexane-
dione (4.0 equiv), piperidine (5.0 equiv), DMF, 5 h; m) ethyl acrylate
(40 equiv), quinuclidine (40 equiv), DMSO, 4 h; n) thiophenol (15 equiv),
TCEP (10 mm), Et3N (5%), DMF/H2O (9:1), 508C, 12 h; o) NH2NH2
(20 equiv), DIPEA (50 equiv), DMSO, 508C, 12 h; p) 2,4-pentanedione
(15 equiv), EtOH, 508C, 12 h; q) chloroquinazoline (5.0 equiv), DIPEA
(7.0 equiv), DMF, 508C, 12 h; r) FmocNCS (3.0 equiv), pyridine
(5.0 equiv), CH2Cl2, 3 h; s) 2-bromoacetophenone (0.1m), dioxane, 2ꢂ
3 h; t) TFA; 12 h; u) benzaldehyde (10 equiv), TMOF, 12 h; TFA,
8 h. HOBt=hydroxybenzotriazole, DIC=N,N’-diisopropylcarbodiimide,
NMP=N-methyl-2-pyrrolidone, TBACl=tetrabutylammonium chloride,
Dppe=1,2-bis(diphenylphosphino)ethane, Grubbs I=benzylidene-bis(tri-
cyclohexylphosphane)dichlororuthenium(IV) dichloride, DCE=1,2-di-
chloroethene, TBTA=tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine,
TMOF=trimethyl orthoformate, DMSO=dimethyl sulfoxide, TCEP=
(tris(2-carboxyethyl)phosphane), TFA=trifluoroacetic acid.
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N. Winssinger et al.
Conclusion
We have developed a synthetic
strategy for PNA-encoded syn-
thesis (PES) that is broadly
compatible with most reaction
prototypes used in library syn-
thesis and DOS. In addition,
we have developed simple and
scalable protocols for Mtt-pro-
tected PNA bearing g-modifi-
cations. PNA-oligomers incor-
porating these modified resi-
dues afford higher sensitivity
and fidelity in hybridization.
These developments should
also be applicable to parallel
efforts using PNA-conjugates
to organize ligands with con-
trolled geometry.[26]
Experimental Section
Full experimental procedures and
spectroscopic characterization data
are provided in the Supporting Infor-
mation.
Figure 5. PES of a b-peptide library.
sources of commercially available bifunctional synthons of
diversity. To illustrate the orthogonality of the Mtt-based
PES with Fmoc-protected synthons, we opted for a library
containing b-amino acids.[26] Such b-amino peptides are
more structured and more resistant towards enzymatic deg-
radation than their counterparts composed of a-amino acids.
As shown in Figure 5, resin 46 was split into five pools and
the Mtt was removed to introduce the first PNA codon (see
the Supporting Information for codon sequences). Although
the use of only five pools per reaction affords a total library
size of 625, which does not fully capitalize on the power of
PES, this restricted library was anticipated to facilitate anal-
ysis. The Fmoc was then removed and the five different
Fmoc protected b-amino acids (Arg, Phe, Glu, Ile, and Ser)
were coupled in their respective pool. The pools were mixed
and split and the cycle of PES was reiterated three times. At
each cycle, an analytical aliquot of the library was cleaved
to evaluate the progression of the synthesis by MALDI MS
analysis. The spectra increase in complexity because the
number of compounds per pool increase from 1 in the first
cycle to 5 then 25 then 125 in the second, third, and forth
cycles, respectively. Nonetheless, the expected molecular
weight window is clearly identified in each cycle with mini-
mal truncation (a capping cycle is used after every coupling,
hence incomplete reaction standout as lower molecular
weight peaks).
Acknowledgements
This work was supported by a grant from the Institut Universitaire de
France (IUF) and the European Research Council (ERC 201749).
MENRT fellowships (M. C., C. Z.) as well as a fellowship from the Al-
gerian Ministry of Higher Education and Scientific Research (DC) are
also gratefully acknowledged.
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Received: April 19, 2012
Revised: June 15, 2012
Published online: && &&, 2012
Chem. Eur. J. 2012, 00, 0 – 0
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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These are not the final page numbers!

N. Winssinger et al.
Combinatorial Chemistry
D. Chouikhi, M. Ciobanu,
C. Zambaldo, V. Duplan, S. Barluenga,
N. Winssinger* ................ &&&& — &&&&
Expanding the Scope of PNA-
Encoded Synthesis (PES): Mtt-
Protected PNA Fully Orthogonal to
Fmoc Chemistry and a Broad Array of
Robust Diversity-Generating Reac-
tions
Keeping track of a library: A strategy
that is compatible with a large palette
of reactions that have been produc-
tively used in diversity oriented syn-
thesis (palladium cross-couplings,
metathesis, p-allylation, CuAAC,
reductive amination, amidation, het-
erocycle formation, nucleophilic addi-
tion, conjugate additions, Pictet–Spen-
gler cyclization) is described (see
figure).
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Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!