On-resin N-terminal peptoid degradation: Toward mild sequencing conditions

Article abstract of DOI:10.1002/bip.22884

A novel approach to sequentially degrade peptoid N-terminal N-(substituted)glycine residues on the solid-phase using very mild conditions is reported. This method relies on the treatment of resin-bound, bromoacetylated peptoids with silver perchlorate in THF, leading to an intramolecular cyclization reaction to liberate the terminal residue as a N-substituted morpholine-2,5-dione, resulting in a truncated peptoid upon hydrolysis and a silver bromide byproduct. Side-chain functional group tolerance is explored and reaction kinetics are determined. In a series of pentapeptoids possessing variable, non-nucleophilic side-chains at the second position (R²), we demonstrate that sequential N-terminal degradation of the first two residues proceeds in 87% and 74% conversions on average, respectively. We further demonstrate that the degradation reaction is selective for peptoids, and represents substantial progress toward a mild, iterative sequencing method for peptoid oligomers.

Full text of DOI:10.1002/bip.22884

OnꢀResin NꢀTerminal Peptoid Degradation: Toward Mild
Sequencing Conditions
Caroline Proulx, Falko Noë, Stan Yoo, Michael D. Connolly, and Ronald N. Zuckermann*
The Molecular Foundry, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley,
California, 94720, United States.
ABSTRACT
A novel approach to sequentially degrade peptoid Nꢀterminal Nꢀ(substituted)glycine
residues on the solidꢀphase using very mild conditions is reported. This method relies on the
treatment of resinꢀbound, bromoacetylated peptoids with silver perchlorate in THF, leading to
an intramolecular cyclization reaction to liberate the terminal residue as a an Nꢀsubstituted
morpholineꢀ2,5ꢀdione, resulting in a truncated peptoid upon hydrolysis and a silver bromide
byproduct. Sideꢀchain functional group tolerance is explored and reaction kinetics are
determined. In a series of pentapeptoids possessing variable, nonꢀnucleophilic side chains at
the second position (R2), we demonstrate that sequential Nꢀterminal degradation of the first two
residues proceeds in 87% and 74% conversions on average, respectively. We further
demonstrate that the degradation reaction is selective for peptoids, and represents substantial
progress toward a mild, iterative sequencing method for peptoid oligomers.
INTRODUCTION
Sequential degradation of polypeptides is an essential analytical tool to determine the
linear sequence of proteins of unknown structure. This sequencing process is typically
achieved using the iterative Edman degradation cycle, which consists of two steps: 1) reaction
of the Nꢀterminal amino group with phenylisothiocyanate (PITC), followed by 2) treatment
with a strong acid (trifluoroacetic acid, TFA), to liberate the Nꢀterminal amino acid fragment as
a phenylthiohydantoin (PTH) (Scheme 1a).1 Partial Edman degradation has also been used to
generate molecular weight ladders that can be analyzed by mass spectrometry for sequence
determination.2 Although the Edman degradation was developed for the sequencing of
polypeptides, it has been applied with good success to the sequencing of Nꢀ(substituted)glycine
peptoid oligomers as well.3,4 The method has found great utility in the identification of hits
from combinatorial peptoid libraries.5,6 However, the Edman degradation conditions utilize
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
‘Accepted Article’, doi: 10.1002/bip.22884
© 2016 Wiley Periodicals, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 2 of 25
relatively harsh, acidic conditions, as well as being limited to peptides ≤50ꢀ60 residues in
length and requiring extended reaction times. As we seek to develop sequencing methods for
peptidomimetic polymer systems, we aim to develop efficient, rapid and mild reaction
conditions, that can be applied generally and with a large diversity of sidechain functionalities.
Here, we report alternate, mild conditions to degrade peptoid polymers, one monomer at a time
starting from the Nꢀterminus (Scheme 1b).
[Scheme 1]
Nꢀterminal peptoid degradation reactions have in fact been previously reported,
although they use harsher conditions and have not been applied iteratively.7 For example, Nꢀ
acylated peptoids have been shown to cleave off the Nꢀterminal residue under acidic cleavage
conditions (TFA), leading to mixtures of the desired Nꢀacylated peptoid and truncated
products.7 This reaction proceeds via an intramolecular cyclization reaction, liberating an
oxazolinium ion and the deletion sequence, which can be suppressed by using electronꢀpoor
acyl groups at the Nꢀterminus (e.g. 4ꢀnitrobenzoyl or trifluoroacetyl groups). In analogy to the
degradation of Nꢀacylated peptoids, Nꢀterminal AcꢀNꢀmethyl amino acid residues and Nꢀ
benzoyl amino acid residues have also been shown to undergo acidꢀpromoted degradation
reactions.8,9 However, in all the abovementioned cases, the cyclization reactions used harsh
conditions, similar to the Edman degradation conditions (TFA), and were dependent on both
the sideꢀchain chemistry of the Nꢀterminal residue and the nature of the acyl group. In addition,
none of these approaches have been explored iteratively for sequencing purposes, with efforts
focusing mainly on suppression of this truncation reaction using electron poor acyl groups to
create libraries with diversity at the Nꢀacylated terminal residue and isolate the desired Nꢀ
acylated peptide or peptoid.
We recently reported a way to accelerate displacement reactions in submonomer
peptoid synthesis using aniline weak nucleophiles, where halophilic silver salts were employed
to promote halide abstraction and formation of an AgBr precipitate.10 In the course of that
work, we found that the treatment of resinꢀbound, bromoacetylated peptoids with AgClO4
solutions in the absence of an amine submonomer leads to onꢀresin truncation of the Nꢀ
terminal residue via an intramolecular cyclization reaction, generating an Nꢀsubstituted
morpholineꢀ2,5ꢀdione byproduct (Scheme 1b). These mild reaction conditions allow the Nꢀ
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 3 of 25
Biopolymers: Peptide Science
terminal degradation of peptoid residues to be performed on acidꢀlabile Rink amide
polystyrene resin, leaving the truncated peptoid bound to solidꢀphase. To evaluate the potential
of this method for sequencing of oneꢀbeadꢀoneꢀcompound (OBOC)11 combinatorial libraries,
we further performed this degradation reaction on Tentagel resin, and report here on the sideꢀ
chain tolerance using a variety of Nꢀsubstituted glycine monomers in a series of chemically
diverse pentapeptoids.
RESULTS AND DISCUSSION
To establish that deletion of an Nꢀterminal residue was occuring, we first demonstrated
that we could remove one Nꢀterminal residue and replace it with another. Thus, tetrapeptoids 2
and 3 were synthesized in parallel, where the Nꢀterminal residue was either a Nꢀ(2ꢀ
methoxyethyl)glycine (Nme) or a Nꢀ(2ꢀphenylethyl)glycine (Npe) residue, respectively (Figure
1a). Nꢀterminal truncation of the Npe residue (3) was accomplished by sequentially treating the
resin with bromoacetic acid and N,N’ꢀdiisopropylcarbodiimide (DIC), followed by AgClO4 in
tetrahydrofuran (THF) for 1 h at room temperature. The resulting truncated peptoid (1’) was
bromoacetylated and treated with a solution of 2ꢀmethoxyethylamine in Nꢀmethylpyrrolidinone
(NMP), and the HPLC retention times of the obtained (corrected) tetrapeptoid (2’) was
compared with that of synthesized tetrapeptoid 2 (Figure 1b). The molar mass (as determined
by electrospray mass spectrometry) and HPLC retention times of both tetramers proved to be
identical, confirming the occurence of a mild and efficient silverꢀmediated, onꢀresin Nꢀterminal
truncation reaction. Interestingly, the resin does not have to be treated with any additional
reagents to hydrolyze the cyclic iminium intermediate (Scheme 1b), which occurs readily
during the washing step with (wet) N,Nꢀdimethylformamide (DMF). Monitoring of the reaction
kinetics by HPLC revealed that complete disappearance of the bromoacetylated peptoid
occured after 1 h, with subsequent DMF wash affording the truncated tripeptoid (1’) (Figure 2).
Of note, the aforementioned reaction was performed on Rink amide polystyrene resin without
any cleavage of the peptoid from the resin. Because the peptoid degradation conditions do not
involve treatment with trifluoroacetic acid (TFA), premature cleavage of the peptoid from solid
support was not observed.
[Figure 1]
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 4 of 25
[Figure 2]
The mechanism of the degradation reaction was confirmed by synthesizing model
peptoid monomer 5. This compound was bromoacetylated to give 6 and treated with silver
perchlorate to afford cyclic product 8 in 78% yield, following the acidꢀcatalyzed hydrolysis of
intermediate 7 (Scheme 2). Compound 8 was verfied to be the Nꢀsubstituted morpholine 2,5ꢀ
dione byproduct by 1H and 13C NMR.
[Scheme 2]
Moreover, collection of the filtrate following the onꢀresin truncation of tetrapeptoid 3 to give
tripeptoid 1’ revealed Nꢀsubstituted morpholine 2,5ꢀdione 8 as the major product by NMR,
following THF evaporation and extraction with 1M HCl / EtOAc (see supporting information).
Using (NmeNpe)4 octapeptoid 9 as starting material, we further demonstrated that the
degradation reaction could be performed iteratively three cycles in a row, affording the desired
truncated peptoids 10ꢀ12 as the major products (Scheme 3 and Figure 3). The reaction
conversions were estimated by measuring the ratio between the crude purities of the truncated
product and starting peptoid, monitored by HPLC at 214 nm, yielding conversions of 86%,
69%, and 76% for cycles 1, 2, and 3, respectively.
[Scheme 3]
[Figure 3]
It is noteworthy that in contrast to the Edman degradation procedure, which liberates a
phenylthiohydantoin byproduct that can be easily detected by HPLC, the lack of a persistent
chromophore on the Nꢀsubstituted morpholineꢀ2,5ꢀdione product released prevents its use in
HPLC analysis for sequencing purposes. However, partial degradations to generate molecular
weight ladders analyzed by mass spectrometry would likely be achievable, similarly to known
partial Edman degradation procedures that have demonstrated utility in sequencing.2
With the desire to employ these newfound mild conditions as an alternative to Edman
degradation conditions for peptoid and peptide sequencing, we synthesized a series of
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 5 of 25
Biopolymers: Peptide Science
hexamers with sideꢀchain diversity on Tentagel MꢀNH2 resin. We designed our test peptoids
13aꢀm to contain diverse sideꢀchain chemistry at the first two positions (R1 and R2), followed
by two Npe monomers and one Nꢀ(2ꢀaminoethyl)glycine (Nae) residue, to facilitate HPLC
detection and mass spectrometry analyses, respectively (Scheme 4 and Table 1). A Cꢀterminal
methionine residue was incorporated to enable CNBrꢀmediated cleavage from solidꢀsupport for
analysis. The Nꢀterminal degradation reactions were monitored by HPLC, where the purity of
the obtained truncated products were compared to the purities of the starting peptoids (Table
1). To enable accurate reaction monitoring by HPLC and mass spectrometry, quenching of
unreacted bromoacetylated peptoid was achieved by treating the resin with a solution of 1.5 M
2ꢀphenylethylamine in DMF prior to cleavage from the solid support. The ratios between the
crude purities of the desired truncated product and starting peptoids are reported in Table 2
(conversions).
We first attempted the Nꢀterminal deletion of Npe and Nme residues (13aꢀb), in order to
determine the efficiency of the reaction using simple, nonꢀnucleophilic sideꢀchains. In these
cases, completion of the degradation reaction was observed after treatment of bromoacetylated
peptoid with AgClO4 for 30 minutes, with the crude purities dropping by only 1ꢀ2% (Table 1,
13aꢀb), indicating a near quantitative Nꢀterminal degradation reaction (Table 2, 13aꢀb). We
next examined the effect of conformationally restricted monomers at both the Nꢀterminal and
second position (R2), such as transꢀinducing Nꢀphenyl glycine (Nph)12 and cisꢀinducing (S)ꢀNꢀ
(1ꢀphenylethyl)glycine (Nspe)13 residues (13cꢀf). The reaction was inhibited (65% conversion)
when the Nph monomer was located at the second position (R2) (Tables 1 and 2, 13c), likely
due to the reduced nucleophilicity of the electronꢀdefficient Nꢀphenyl glycine residue. Of note,
analysis of Nphꢀterminated peptoid was complicated by air oxidation and degradation of the
peptoid over time.10 As expected, deletion of the transꢀinducing Nph residue was sluggish,
providing the desired truncated tetrapeptoid in only 17% conversion (Tables 1 and 2, 13d). A
24% drop in crude purity was observed for the peptoid containing a Nspe residue at the second
position (R2) (Tables 1 and 2, 13e), where the sterically encumbered cisꢀinducing Nspe residue
might be preventing efficient formation of the cyclic intermediate. In contrast, deletion of an
Nspe monomer at the Nꢀterminal position (R1) proceeded smoothly, affording the desired
tetramer in 95% conversion (Table 2, 13f).
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 6 of 25
In order to study the sideꢀchain functional group tolerance of the silverꢀpromoted
deletion reaction more in depth, peptoids 13gꢀl were prepared (Scheme 4a and Table 1), where
the sideꢀchain chemistry at the second position (R2) was varied, and an Nꢀterminal Nme residue
was incorporated (R1). Performing two consecutive deletion reactions provided insight on the
ability of each Nꢀsubstituted glycine to be deleted, as well as their effect on the degradation of
the preceeding Nme residue. On average, the Nꢀterminal Nme residue of peptoids 13gꢀl was
degraded with only 12% loss in crude purity (Table 1) to give tetrapeptoids 14gꢀl in 87%
conversion (Table 2). This indicates that the nature of the preceeding residue (R2), unless it is a
electronꢀpoor residue (e.g. Nph) or sterically encumbered monomer (e.g. Nspe), has little effect
on the efficiency of the onꢀresin truncation. In most cases, the drop in purity was due to the
appearance of a combination of many minor side products, whose structures could not be easily
inferred from mass spectrometry analysis (see supporting information for representative HPLC
traces).
[Scheme 4]
[Table 1]
[Table 2]
We next tried to perform a second consecutive Nꢀterminal degradation reaction cycle. In this
case, the bromoacetylation step was performed 2 x 20 minutes in order to ensure that the AgBr
precipitate from the first degradation cycle was fully dissolved and washed away.10 Following
treatment of the resinꢀbound bromoacetylated tetrapeptoids with silver perchlorate to give
tripeptoid 15, a drop of ~23% in purity of was observed on average (Table 1). Nonetheless, in
most cases, the desired truncated tripeptoid 15 was obtained as the major product in >50%
crude purity, with an average conversion of 74% to the desired truncated peptoid (Table 2). As
a notable exception, degradation of Ncpl residue in tetrapeptoid 14g only resulted in 27%
conversion to 15 after treatment of bromoacetylated peptoid with AgClO4 for 1 h, indicative of
slower reaction kinetics with an Nꢀcyclopropyl side chain.
We next attempted to perform Nꢀterminal degradation reactions on more challenging
substrates, containing a nucleophilic heteroatom in the sideꢀchain (Scheme 4b, 13mꢀn). In
these cases, the resinꢀbound peptoids were first treated with TFA for 10 minutes to remove of
the tertꢀbutyloxycarbonyl (Boc) and tetrahydropyranyl acetal (THP) protecting groups,
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 7 of 25
Biopolymers: Peptide Science
respectively, followed by exposure to the degradation cycle conditions as above. Peptoid 13m
is structurally similar to peptoid 13a, where the Boc protecting group on the Nae residue has
been removed. The first step presumably leads to bromoacetylation at both the Nꢀterminal Nꢀ
substituted glycine and the amino sideꢀchain functional group, which raises the question of
how a bromoacetylated side chain might react upon treatment with the halophilic silver salt. In
attempting to degrade the Nꢀterminal Nme residue in peptoid 13m, tetrapeptoid 14m was
obtained as the major product in 60% purity after quenching with 2ꢀphenylethylamine (Scheme
4b), corresponding to a 32% drop in purity compared to the starting peptoid (Table 1). Despite
obtaining the expected truncated peptoid as the major product, a significant increase in the
number of side products formed was observed compared to peptoid analog 13a, where less
than 1% drop in purity was observed when the Nae side chain was Bocꢀprotected. The structure
of the multiple minor side products could not be easily determined by mass spectrometry
analysis. Similarly, performing two sequential degradation reactions on peptoid 13n possessing
both an unprotected Nser residue at the second position (R2) and unprotected Nae residue
yielded multiple side products and afforded the desired truncated trimer in less than 40% purity
(Table 1).
In addition to the aforementioned challenging substrates, we attempted to perform the
degradation reaction in peptoidꢀpeptide hybrids 13oꢀq, where the second residue was either an
alanine, phenylalanine, or proline amino acid (Scheme 5). Degradation of the Nꢀterminal Nme
residue proceeded in 76ꢀ84% conversions (Table 4, 13oꢀq) to give peptideꢀpeptoid hybrids
14oꢀq, revealing that amino acid residues do not inhibit formation of the sixꢀmembered cyclic
intermediate when located at the second position (R2). However, degradation of the amino
acids proved to be unsucessful in all three cases, giving unreacted bromoacetylated Nꢀterminal
amino acids exclusively, as detected by mass spectrometry following quenching with an amine
submonomer. In order to rule out the possibility that steric interactions prevented amino acids
residues to be deleted in peptideꢀpeptoid hybrids, a test peptide sequence was synthesized to
evaluate its ability to be degraded using our conditions. Specifically, tetrapeptide PheꢀPheꢀLysꢀ
Met was subjected to our degradation conditions following Fmoc removal. Once again,
deletion of the Nꢀterminal amino acid (Phe) proved to be unsuccessful, reiterating that amino
acid residues cannot be degraded by this mechanism, and that the degradation reaction is
selective for peptoids.
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 8 of 25
[Scheme 5]
[Table 3]
[Table 4]
CONCLUSION
In summary, we demonstrated that Nꢀsubstituted glycine peptoid monomers can be
degraded sequentially from the Nꢀterminus using novel, mild conditions that avoid treatment
with strong acids. Specifically, resinꢀbound peptoids are (1) bromoacetylated and (2) treated
with a silver salt, thereby promoting an intramolecular cyclization reaction that leads to
removal of the Nꢀterminal residue as an Nꢀsubstituted morpholineꢀ2,5ꢀdione and a free Nꢀ
terminus. The identity of the morpholineꢀ2,5ꢀdione byproduct was confirmed via solutionꢀ
phase synthesis, isolation and characterization of a model 4ꢀ(2ꢀphenylethyl)morpholineꢀ2,5ꢀ
dione compound (8). Using a series of pentapeptoids with variable, nonꢀnucleophilic side
chains at the second position (R2), we demonstrated that two sequential degradation reactions
could be performed in 87% and 74% conversions on average, respectively. Amino acids,
however, could not be truncated using these conditions, indicating that this reaction is selective
for peptoids. For resinꢀbound peptoids with protected sideꢀchains, the mild conditions for Nꢀ
terminal degradation described here may find utility in the activityꢀbased editing of peptoids,
where inactive compounds identified from OBOC library screening may be subjected to
degradation of the Nꢀterminal residue, followed by addition of a new monomer. Further
developments will aim to improve the conversions yields of each degradation cycle to allow
sequencing of longer peptoid oligomers.
EXPERIMENTAL SECTION
General. Solidꢀphase chemistry was performed using polystyrene Rink Amide resin (0.57
mmol/g) or Tentagel MꢀNH2 resin (0.3 mmol/g) in filtration tubes equipped with caps and
stopcocks. Analytical HPLC analyses were performed on a 5 ꢁm, 150 mm x 4.6 mm C18
Vydac columnTM at 60 °C with a flow rate of 1.0 mL/min using a 20ꢀ80% gradient, where
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 9 of 25
Biopolymers: Peptide Science
solvent A = water [0.1% trifluoroacetic acid (TFA)] and solvent B = MeCN (0.1% TFA).
LCMS analyses were performed on a 5 ꢁm, 50 mm x 2.1 mm C18 Vydac column with a flow
rate of 0.35 mL/min using 3ꢀ100% gradient. HPLC traces were monitored at 214 nm.
Analytical thinꢀlayer chromatography (TLC) was performed on glassꢀbacked silica gel plates.
Visualization of the developed chromatogram was performed by UV absorbance. Silica gel
chromatography was performed using an SP1 Flash Chromatography instrument from Biotage.
Nuclear magnetic resonance spectra (1H, 13C) were recorded on a Bruker AV300.
Reagents. Bromoacetic acid, N,N’ꢀdiisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA),
triisopropylsilane
(TIPS),
Oꢀ(Benzotriazolꢀ1ꢀyl)ꢀN,N,N’N’ꢀtetramethyluronium
hexafluorophosphate (HBTU), N,Nꢀdiisopropylethylamine (DIEA), cyanogen bromide, all
amine and amino acid reagents were purchased from commercial sources and used without
further purification. Silver perchlorate was dried by azeotrope evaporation with benzene and
placed in a desiccator under vacuum with P2O5 to give a white solid that was stored in a
desiccator wrapped with foil. Solvents were purchased from commercial sources.
Synthetic Experimental Procedures. Peptoids and peptoidꢀpeptide hybrids 13aꢀq were
synthesized according to standard solidꢀphase procedures.14
2ꢀBromoꢀ1ꢀmorpholinoethanꢀ1ꢀone (4)
Morpholine (0.43 mL, 4.95 mmol) was dissolved in 25 mL of dry DCM in a dry 50 mL flask,
treated with triethylamine (0.76 mL, 5.45 mmol), and cooled to 0°C. Bromoacetyl bromide was
added dropꢀwise at 0°C, and the reaction mixture was allowed to warm to room temperature
overnight. The solution was placed in a separatory funnel and the organic phase was washed
sequentially with H2O (25 mL), 5% citric acid (25 mL), followed by saturated sodium
bicarbonate (25 mL). The organic phase was dried over Na2SO4, filtered and evaporated under
reduced pressure. The crude oil was purified by silica gel chromatography using an SP1 Flash
Chromatography instrument from Biotage with a gradient from 60 to 70% ethyl acetate in
hexanes. Evaporation of the collected fractions afforded 2ꢀbromoꢀ1ꢀmorpholinoethanꢀ1ꢀone 4
1
as a yellow oil (886 mg, 86% yield): Rf 0.34 (1:1 EtOAc: hexanes). H NMR (500 MHz,
CDCl3) δ 3.40 (2H, t, J = 5.0 Hz), 3.47 (2H, t, J = 5.0 Hz), 3.55 (2H, t, J = 4.5 Hz), 3.60 (2H, t,
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 10 of 25
13
J = 5.0 Hz), 3.76 (2H, s). C NMR (125 MHz, CDCl3) δ 165.3, 66.4, 66.3, 47.0, 42.3, 25.7.
MS m/z 208, (M+H)+ calcd for [C6H11BrNO2]+ : 208.0.
1ꢀMorpholinoꢀ2ꢀ(phenethylamino)ethanꢀ1ꢀone (5)
2ꢀBromoꢀ1ꢀmorpholinoethanꢀ1ꢀone 4 (1 g, 4.83 mmol) was dissolved in 25 mL of dry THF in a
dry 50 mL flask, cooled to 0°C, and treated sequentially with triethylamine (1.35 mL, 9.66
mmol) and phenylethylamine (2.43 mL, 19.32 mmol) at 0°C. The solution was allowed to
warm to room temperature and stirred overnight. Reaction mixture was filtered, THF was
removed in vacuo, and the crude oil was purified using an SP1 Flash Chromatography
instrument from Biotage with a gradient from 1 to 6 % methanol in dichloromethane.
Evaporation of the collected fractions afforded 1ꢀmorpholinoꢀ2ꢀ(phenethylamino)ethanꢀ1ꢀone 5
as a dark yellow oil (1.2 g, >99% yield): Rf 0.22 (10% MeOH in DCM). 1H NMR (500 MHz,
CDCl3) δ 2.83ꢀ2.86 (2H, m), 2.89ꢀ2.93 (2H, m), 3.40 (2H, t, J = 5.0 Hz), 3.44 (2H, s), 3.62ꢀ
13
3.70 (6H, m), 7.21ꢀ7.33 (5H, m). C NMR (125 MHz, CDCl3) δ 169.7, 139.9, 128.7, 128.5,
126.2, 66.9, 66.5, 51.4, 50.5, 45.0, 42.1, 36.7. MS m/z 249, (M+H)+ calcd for [C14H21N2O2]+ :
249.2.
2ꢀBromoꢀNꢀ(2ꢀmorpholinoꢀ2ꢀoxoethyl)ꢀNꢀphenethylacetamide (6)
1ꢀMorpholinoꢀ2ꢀ(phenethylamino)ethanꢀ1ꢀone 5 (1.2 g, 4.83 mmol) was dissolved in 25 mL of
dry THF in a dry 50 mL flask, cooled to 0°C, and treated sequentially with triethylamine (808
ꢁL, 5.80 mmol) and bromoacetyl bromide (505 ꢁL, 5.80 mmol), and stirred at 0°C for 1 h. The
volatiles were removed on a rotary evaporator under reduced pressure to give a residue, which
was purified using an SP1 Flash Chromatography instrument from Biotage with a gradient
from 60 to 100 % ethylacetate in hexanes. Evaporation of the collected fractions afforded 2ꢀ
bromoꢀNꢀ(2ꢀmorpholinoꢀ2ꢀoxoethyl)ꢀNꢀphenethylacetamide 6 as an orange solid (840 mg,
48%): Rf 0.16 (8:2 EtOAc: hexanes). 1H NMR (500 MHz, CDCl3) Major rotamer: δ 2.99 (2H,
t, J = 7.0 Hz), 3.46 (2H, t, J = 4.8 Hz), 3.59ꢀ3.64 (2H, m), 3.66 (2H, s), 3.70ꢀ3.75 (6H, m), 4.15
(2H, s), 7.22ꢀ7.36 (5H, m). Minor rotamer: 2.91 (2H, t, J = 7.0 Hz), 3.22 (2H, t, J = 4.9 Hz),
3.62 (2H, 2), 3.59ꢀ3.64 (2H, m), 3.70ꢀ3.75 (6H, m), 3.87 (2H, s), 7.22ꢀ7.36 (5H, m). 13C NMR
(125 MHz, CDCl3) (mixture of rotamers) δ 167.6, 167.3, 166.1, 165.9, 139.2, 137.9, 129.0,
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 11 of 25
Biopolymers: Peptide Science
128.8, 128.7, 128.6, 127.0, 126.5, 66.8, 66.5,66.2, 51.2, 50.5, 50.4, 46.9, 45.4, 45.0, 42.5, 42.3,
34.7, 33.9, 26.7, 25.6. MS m/z 369, (M+H)+ calcd for [C16H22BrN2O3]+ : 369.1.
4ꢀ(2ꢀPhenylethyl)morpholineꢀ2,5ꢀdione (8)
2ꢀBromoꢀNꢀ(2ꢀmorpholinoꢀ2ꢀoxoethyl)ꢀNꢀphenethylacetamide 6 (54 mg, 0.145 mmol) was
dissolved in 2 mL of dry THF in a dry 5 mL flask, treated with AgClO4 (38 mg, 0.183 mmol)
and stirred for 1 h. 1 mL of a 1M HCl solution was added to the flask, and the reaction mixture
was left stirring for 30 minutes. The solution was filtered and the volatiles were removed under
reduced pressure. EtOAc was added and the organic phase was washed with brine (3 x 5 mL),
dried over Na2SO4, filtered, and evaporated under reduced pressure to give 4ꢀ
phenethylmorpholineꢀ2,5ꢀdione 8 as a white solid (23 mg, 78%): Rf 0.36 (8:2 EtOAc:
hexanes). 1H NMR (500 MHz, CDCl3) δ 2.94 (2H, t, J = 7.0 Hz), 3.68 (2H, t, J = 7.0 Hz), 3.89
13
(2H, s), 4.74 (2H, s), 7.22ꢀ7.37 (5H, m). C NMR (125 MHz, CDCl3) δ 164.7, 163.3, 137.7,
129.0, 128.7, 127.1, 67.7, 48.6, 48.2, 33.4. MS m/z 220, (M+H)+ calcd for [C12H14NO3]+ :
220.1.
Representative procedure for silver perchlorateꢀmediated degradation reaction of
peptoid monomers. To 20 mg of resinꢀbound peptoid 13aꢀq in a 1 mL fritted cartridge was
added 500 ꢁL of DMF. Resin was allowed to swell for 20 minutes before draining. 500 ꢁL of a
0.6 M solution of bromoacetic acid and 43 ꢁL (0.92 equiv, 0.28 mmol) of DIC was added and
allowed to react for 20 minutes. The resin was washed with DMF (3 x 1 mL) and THF (2 x 1
mL) before adding 500 ꢁL of a 36 mM stock solution (3 equivalents) of AgClO4 in THF. The
reaction was allowed to proceed for 0.5 to 1 h, after which the reagents were drained and the
resin was washed with DMF (3 x 1 mL) and DCM (1 x 1 mL). For reaction monitoring, a small
amount of resin was treated with 500 ꢁL 1.5 M solution of 2ꢀphenylethylamine in DMF for 10
minutes to quench any unreacted bromoacetylated peptoids. The resin was drained and washed
with DMF (3 x 1 mL) and DCM (1 x 1 mL), followed by treatment with a 95:5 TFA:H2O v/v
mixture for 10 minutes to ensure complete deprotection of Bocꢀprotected sideꢀchains. The resin
was washed with DCM (3 x 1 mL), treated with 100 ꢁL of 2 mg/mL of CNBr in MeCN:0.25 N
HCl 1:1, covered with aluminum foil, and allowed to react for 12ꢀ16 h. The cleavage solution
was collected by filtering the resin through a disposable, polypropylene fritted cartridge into a
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 12 of 25
1.5 mL Eppendorf tube. The filtrate was evaporated to dryness under vacuum using a
SpeedVac concentrator, and reꢀdissolved in 1:1 MeCN:H2O prior to analysis by HPLC and
LC/MS.
ACKNOWLEDGMENTS
This work was supported by the Defense Threat Reduction Agency under Contract No.
DTRA10027ꢀ15875 and the DARPA Fold F(x) program. The work was conducted at the
Molecular Foundry with support by the Office of Science, Office of Basic Energy Sciences,
U.S. Department of Energy, under Contract No. DEꢀAC02ꢀ05CH11231. C. P. is grateful for a
postdoctoral fellowship from the Natural Sciences and Engineering Council of Canada
(NSERC).
REFERENCES
1. Edman, P. Acta Chem. Scand. 1950, 4, 283.
2. (a) Wang, P.; Arabaci, G.; Pei, D. J. Comb. Chem. 2001, 3, 251. (b) Sweeney, M. C.; Pei, D.
J. Comb. Chem. 2003, 5, 218. (c) Thakkar, A.; Wavreille, A. S.; Pei, D. H. Anal. Chem. 2006,
78, 5935.
3. Thakkar, A.; Cohen, A. S.; Connolly, M. D.; Zuckermann, R. N.; Pei, D. J. Comb. Chem.
2009, 11, 294.
4. Sun, J.; Zuckermann, R. N. ACS Nano 2013, 7, 4715.
5. Kodadek, T.; BachhawatꢀSikder, K. Mol. Biosyst. 2006, 2, 25–35.
6. Reddy, M. M.; BachhawatꢀSikder, K.; Kodadek, T. Chem. Biol. 2004, 11, 1127–1137.
7. Kim, S.; Biswas, G.; Park, S.; Kim, A.; Park, H.; Park, E.; Kim, J.; Kwon, Y. U. Org.
Biomol. Chem. 2014, 12, 5222.
8. Fang, W. J.; Bennett, M. A.; Aldrich, J. V. Biopolymers 2011, 96, 97.
9. Samaritoni, J. G.; Copes, A. T.; Crews, D. K.; Glos, C.; Thompson, A. L.; Wilson, C.;
O'Donnell, M. J.; Scott, W. L. J. Org. Chem. 2014, 79, 3140.
10. Proulx, C.; Yoo, S.; Connolly. M. D.; Zuckermann, R. N. J. Org. Chem. 2015, 80, 10490.
11. Lam, K. S., Lebl, M., and Krchnák, V. Chem. Rev. 1997, 97, 411.
12. Shah, N. H.; Butterfoss, G. L.; Nguyen, K.; Yoo, B.; Bonneau, R.; Rabenstein, D. L.;
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 13 of 25
Biopolymers: Peptide Science
Kirshenbaum, K. J. Am. Chem. Soc. 2008, 130, 16622−16632.
13. For a selected example, see: Wu, C. W.; Sanborn, T. J.; Huang, K.; Zuckermann, R. N.;
Barron, A. E. J. Am. Chem. Soc. 2001, 123, 6778−6784.
14. Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am. Chem. Soc. 1992, 114,
10646−10647.
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 14 of 25
Scheme 1. (a) Edman degradation conditions1 vs (b) a mild approach for N-terminal degradation using
silver salts.
150x148mm (300 x 300 DPI)
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 15 of 25
Biopolymers: Peptide Science
Figure 1. (a) AgClO4-promoted N-terminal deletion of a N-(2-phenylethyl)glycine residue followed by
peptoid elongation. (b) HPLC trace of tetrapeptoid (2) and corrected tetrapeptoid (2’) co-injected.
194x163mm (300 x 300 DPI)
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 16 of 25
Figure 2. Progress of the N-terminal degradation reaction (% conversion) vs time of peptoid 3 to yield 1’.
131x99mm (300 x 300 DPI)
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 17 of 25
Biopolymers: Peptide Science
Scheme 2. Synthesis of model bromoacetylated peptoid 6 and silver-promoted formation of N-substituted
morpholine 2,5-dione product 8.
160x101mm (300 x 300 DPI)
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 18 of 25
Scheme 3. Iterative silver-promoted N-terminal truncations of octapeptoid 9 to afford pentapeptoid 12
after three cycles.
212x108mm (300 x 300 DPI)
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 19 of 25
Biopolymers: Peptide Science
Figure 3. HPLC chromatograms of starting peptoid 9 and truncated peptoids 10-12 following one, two, and
three silver-promoted truncation reactions, respectively.
221x83mm (300 x 300 DPI)
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 20 of 25
Scheme 4. Sequential AgClO4-promoted degradation reactions conducted with peptoids 13a-n, where (a)
the amino nucleophilic side chain of the Nae residue is Boc-protected, and (b) the nucleophilic side chains
(Nae, Nser) are deprotected prior to degradation cycle.
323x264mm (300 x 300 DPI)
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 21 of 25
Biopolymers: Peptide Science
Table 1. Crude purity, mass, and retention time of pentamers, tetramers, and trimers following 1
or 2 subsequent N-terminal silver-promoted degradation reactions of the pentamers.
Peptoid
Crude
purity
of
5mers
(%)
RT of
5mers
(min)a
m/z obsd
(calcd) of
5mers
[M + 1]+
Crude
purity of 4mers
4mers
14aꢀn
RT of
m/z obsd
(calcd) of
4mers
Crude
purity of
3mer 15
(%)
14aꢀn
(min)a
11a-n
[M + 1]+
(%)
13a: R1 = Nme, R2 = Nme
13b: R1 = Npe, R2 = Nme
13c: R1 = Nme, R2 = Nph
13d: R1 = Nph, R2 = Nme
13e: R1 = Nme, R2 = Nspe
13f: R1 = Nspe, R2 = Nme
13g: R1 = Nme, R2 = Ncpl
13h: R1 = Nme, R2 = Ncyh
13i: R1 = Nme, R2 = Ntyr
13j: R1 = Nme, R2 = Nbz
13k: R1 = Nme, R2 = Nleu
13l: R1 = Nme, R2 = Nbsa
13me: R1 = Nme, R2 = Nme
13ne: R1 = Nme, R2 = Nser
92
88
95
71
97
96
97
96
92
96
90
81
92
86
7.71
755.0 (753.4)
800.8 (799.4)
92
86
62
12
73
91
88
84
81
72
82
74
60
<67
6.60
6.63
28.07 b
639.9 (639.3)
n/a
n/a
n/a
n/a
n/a
n/a
24
10.55
640.3 (639.3)
- c(657.3)
24.88 b 773.1 (772.4)
27.64b 774.2 (772.4)
19.84 b 663.0d (639.3)
11.03
10.05
7.79
801.2 (800.4)
801.2 (800.4)
737.4 (736.4)
779.5 (778.4)
818.0 (816.4)
787.3 (786.4)
753.4 (752.4)
880.7 (879.4)
754.7 (754.4)
741.4(740.4)
9.79
6.67
6.44
9.07
9.19
8.88
8.00
6.75
9.77
7.39
686.1 (685.4)
640.1 (639.3)
622.1 (621.3)
664.6 (663.4)
702.6 (701.4)
674.6 (671.4)
638.2 (637.4)
784.0 (764.3)
802.1 (800.4)
788.3(786.4)
9.93
77
10.28
10.33
9.28
63
56
64
7.55
66
7.71
n/a
<38
5.65
aAnalytical HPLC analyses were performed on a Vydac column (4.6 mm x 150 mm, 5 µm, C18) at 60 °C with a
flow rate of 1.0 mL/min using a 20−80% gradient of CH3CN (0.1% TFA) in water (0.1% TFA) over 20 min. HPLC
traces were monitored at 214 nm.
bAnalytical HPLC analyses were performed as above, using a 5−40% gradient of CH3CN (0.1% TFA) in water
(0.1% TFA) over 20 min.
c Mass was not observed due to degradation of Npe-terminated peptoids.
d Observed mass corresponds to sodium adduct, [M + Na]+
eResin was treated with TFA for side-chain protecting group removal, followed by bromoacetylation and Ag-
mediated degradation reaction.
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 22 of 25
Table 2. % Conversions for the first and second degradation reactions
Peptoid
% Conversiona
% Conversiona
for degradation 1
for degradation 2
13a: R1 = Nme, R2 = Nme
13b: R1 = Npe, R2 = Nme
13c: R1 = Nme, R2 = Nph
13d: R1 = Nph, R2 = Nme
13e: R1 = Nme, R2 = Nspe
13f: R1 = Nspe, R2 = Nme
13g: R1 = Nme, R2 = Ncpl
13h: R1 = Nme, R2 = Ncyh
13i: R1 = Nme, R2 = Ntyr
13j: R1 = Nme, R2 = Nbz
13k: R1 = Nme, R2 = Nleu
13l: R1 = Nme, R2 = Nbsa
13mb: R1 = Nme, R2 = Nme
13nb: R1 = Nme, R2 = Nser
>99
98
65
17
75
95
91
88
88
n/a
n/a
n/a
n/a
n/a
n/a
27
92
78
78
78
75
91
91
89
65
n/a
<78c
<57c
a% conversion = [(%purity of degraded peptoid) / (% purity of starting peptoid)]
x 100.
b Resin was treated with TFA for side-chain protecting group removal, followed
by bromoacetylation and Ag-mediated degradation reaction.
c
HPLC analysis was hindered by the presence of multiple peaks near the
injection peak.
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 23 of 25
Biopolymers: Peptide Science
Scheme 5. Sequential AgClO4-promoted degradation reactions conducted with peptoid-peptide hybrids
13o-q.
133x54mm (300 x 300 DPI)
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science
Page 24 of 25
Table 3. Crude purity, mass, and retention time of peptoid-peptide hybrid pentamers and
tetramers following N-terminal silver-promoted degradation reactions of Nme residue.
Peptoid
Crude
purity
of
5mers
(%)
RT of
5mers
(min)a
m/z obsd
(calcd) of
5mers
[M + 1]+
Crude
purity of 4mers
4mers
14nꢀp
RT of
m/z obsd
(calcd) of
4mers
14nꢀp
(min)a
14a-n
[M + 1]+
(%)
13o: R1 = Nme, R2 = Me (Ala)
13p: R1 = Nme, R2 = CH2C6H5 (Phe)
13q: R1 = Nme, R2 = Pro
90
87
81
6.12
8.77
6.39
711.1 (710.9)
787.2 (786.9)
737.1 (736.9)
71
67
68
5.60
8.40
5.89
596.6 (595.7)
672.0 (671.8)
622.0(621.8)
aAnalytical HPLC analyses were performed on a Vydac column (4.6 mm x 150 mm, 5 µm, C18) at 60 °C with a
flow rate of 1.0 mL/min using a 20−80% gradient of CH3CN (0.1% TFA) in water (0.1% TFA) over 20 min. HPLC
traces were monitored at 214 nm.
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.

Page 25 of 25
Biopolymers: Peptide Science
Table 4. % Conversions for the first degradation reactions of amino acid-containing peptide-
peptoid hybrid pentamers
Peptoid
% Conversiona
for degradation 1
13o: R1 = Nme, R2 = Me (Ala)
13p: R1 = Nme, R2 = Ph (Phe)
13q: R1 = Nme, R2 = Pro
79
76
84
a% conversion = [(% purity of degraded peptoid) / (% purity of starting peptoid)] x 100.
John Wiley & Sons, Inc.
This article is protected by copyright. All rights reserved.