Submonomer synthesis of peptoids containingtrans-inducingN-imino- andN-alkylamino-glycines

Article abstract of DOI:10.1039/d1sc00717c

The use of hydrazones as a new type of submonomer in peptoid synthesis is described, giving access to peptoid monomers that are structure-inducing. A wide range of hydrazones were found to readily react with α-bromoamides in routine solid phase peptoid submonomer synthesis. Conditions to promote a one-pot cleavage of the peptoid from the resin and reduction to the correspondingN-alkylamino side chains were also identified, and both theN-imino- andN-alkylamino glycine residues were found to favor thetrans-amide bond geometry by NMR, X-ray crystallography, and computational analyses.

Full text of DOI:10.1039/d1sc00717c

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Submonomer synthesis of peptoids containing
trans-inducing N-imino- and N-alkylamino-
glycines†
Cite this: Chem. Sci., 2021, 12, 8401
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Carolynn M. Davern, Brandon D. Lowe, Adam Rosfi, Elon A. Ison
*
and Caroline Proulx
The use of hydrazones as a new type of submonomer in peptoid synthesis is described, giving access to
peptoid monomers that are structure-inducing. A wide range of hydrazones were found to readily react
with a-bromoamides in routine solid phase peptoid submonomer synthesis. Conditions to promote
a one-pot cleavage of the peptoid from the resin and reduction to the corresponding N-alkylamino side
chains were also identified, and both the N-imino- and N-alkylamino glycine residues were found to
favor the trans-amide bond geometry by NMR, X-ray crystallography, and computational analyses.
Received 5th February 2021
Accepted 9th May 2021
DOI: 10.1039/d1sc00717c
rsc.li/chemical-science
continue expanding the toolbox of trans-amide inducing pep-
toid monomers.
Introduction
In solid-phase submonomer peptoid synthesis, cycles of (1)
acylation with activated bromoacetic acid and (2) SN2
displacement with primary amines are repeated until the
desired peptoid length is achieved (Scheme 1).11 Optimization is
oen required to install structure-inducing residues, which
tend to be sterically hindered and less nucleophilic. This
includes aniline nucleophiles used in the synthesis of N-aryl
glycines, the most widely utilized trans-inducing peptoid
monomers to date. To improve the synthesis of peptoids pos-
sessing multiple N-aryl glycines, a method relying on the addi-
tion of silver perchlorate during the displacement step with
electron-poor anilines was recently developed.12 However,
given the importance of both side chain diversity and secondary
structure rigidication in peptidomimicry, novel trans-amide
inducing peptoid residues that are amenable to rapid auto-
mated library synthesis using standard procedures would be
benecial. Moreover, the discovery of peptoid monomers that
can reinforce side chain-to-side chain intermolecular
Peptoids are N-substituted glycine oligomers that have been
widely pursued to mimic both peptides and synthetic polymers
due to their increased stability, ease of synthesis, and large side
chain diversity.1 Compared to peptides, peptoids maintain the
same glycine backbone; however, the side chain is located on
the nitrogen atom, resulting in tertiary amides devoid of ster-
eocenters and hydrogen bond donors (NH). To counter the
resulting increase in backbone exibility, a variety of peptoid
side chains have been developed that induce either the cis- or
trans-amide geometry through steric and electronic effects. For
example, N-substituted glycines with a-chiral benzylic,2 tert-
butyl/a-gem-dimethylated,3 triazolium,4 uorinated,5 and
cationic alkyl ammonium6 side chains have all been found to
favor the cis-amide bond conguration (Fig. 1a). Comparatively
fewer options exist to reinforce trans-amide bonds, which
include N-(aryl)-,2c-e,7 N-(alkoxy)-,8 N-(hydroxyl)-,9 and N-(acyl
hydrazide)10 glycines (Fig. 1b). With this existing set of
conformationally-restricted peptoid monomers, several
secondary structures such as helices, ribbons, loops, turns, and
sheet-like structures have been accessed,1 making peptoids
a versatile, structurally tunable platform to design folded
structures. However, poor water solubility and limited func-
tional group diversity plague many of the existing structure-
inducing peptoid monomers, and there remains a need to
Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204,
USA. E-mail: cproulx@ncsu.edu
† Electronic supplementary information (ESI) available: Experimental procedures;
LCMS analytical data; NMR spectra. CCDC 2061173, 2061174, 2061175, 2061176,
2061177 and 2061240. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/d1sc00717c
Fig. 1 N-Substituted glycine (peptoid) monomers that favor (a) the cis
or (b) trans-amide bond geometry.
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of a new side chain – backbone hydrogen bond (Fig. 2) and
changes in the side chain dihedral angles (c) upon reduction.
Results and discussion
Solid phase submonomer synthesis and resin cleavage
To validate the use of hydrazones as submonomers and verify
compatibility with other peptoid monomers, sandwich
sequences15 were rst synthesized, alternating between benz-
aldehyde hydrazone as a representative example and a set of
eight other primary amines (Scheme 1). We included N-phenyl
glycine in our study in attempts to afford all-trans peptoid
oligomer 4h. In all cases, the displacement step was performed
by treating the resin-bound bromoacetylated peptoid with
a 1.5 M solution of amine or hydrazone in DMF for 1 h, which
are standard conditions for solid phase submonomer peptoid
synthesis. Most analogs were obtained in high conversions,
demonstrating the ease of using benzaldehyde hydrazone as
a submonomer. Notably, there was no evidence of trans-imi-
nation reactions upon repeated treatment with excess primary
amines.‡ However, using standard procedures, 4h was only
detected in ꢀ20% conversion by LCMS analysis. While it is
possible that the N-terminal N-aryl glycine oxidized,12,16 the
corresponding a-oxo aldehyde side product was not observed
and we attributed the low crude purity to difficulties in the
bromoacetylation of non-nucleophilic N-aryl glycine residue-
s.7a,12,16c This demonstrates that N-imino glycines are easier to
incorporate than commonly employed trans-inducing N-aryl
glycine peptoid monomers.
N-Imino glycines were found to undergo undesired acid-
catalyzed hydrolysis during cleavage from the resin using
95 : 5 TFA : H2O, giving unsubstituted N-amino glycine resi-
dues. However, in the case of benzaldehyde hydrazone, this side
reaction was only prevalent when the N-imino glycine residue
was at the N-terminus; acetylation or embedding this residue
within a peptoid resulted in little to no side chain hydrolysis for
peptoids 4a–h (ESI†). Reduction of the C]N bond was also
observed when common carbocation scavengers [e.g. triisopro-
pylsilane (TIPS) and triethylsilane (TES)] were added to the
cleavage cocktail. Hydrazone reduction during resin cleavage
could provide rapid access to valuable peptoid monomers with
a new hydrogen bond donor site (NH), eliminating the need for
subsequent treatment with sodium cyanoborohydride. We
hypothesized that both N-imino- and N-alkylamino glycine-
containing peptoids could be obtained cleanly contingent on
the cleavage cocktail used. Thus, careful examination of
different resin cleavage conditions was undertaken using tri-
peptoid 5 as a model compound (Table 1). Using a standard
cleavage cocktail of 95 : 2.5 : 2.5 TFA : H2O : TIPS resulted in
a mixture of products and low crude purity aer 2 h, and should
therefore be avoided (Table 1, entries 1 and 2). However,
cleavage of the resin-bound peptoid using 95 : 5 TFA : H2O in
the absence of TIPS provided compound 6 as the major product
(Table 1, entries 3 and 4), and using 95 : 5 TFA : TES without
water afforded the reduced peptoid 7 in good crude purities
(Table 1, entries 5 and 6).
Scheme 1 Crude purities of peptoid pentamers 4a–h. LCMS purity of
the crude peptoid at 214 nm. Unless otherwise noted, peptoids were
cleaved from the resin using 95 : 5 TFA : H2O for 10 minutes.
a
Cleavage was performed using 95 : 5 TFA : H2O for 2 h.
interactions (e.g. p stacking, hydrogen-bonding) while also
restricting backbone dihedral angles could be impactful in
nanomaterials applications.13 Hydrazide submonomers have
been explored to expand side chain diversity, introduce
hydrogen bond donors/acceptors in the side chain, and rein-
force trans-amide bond conformation (Fig. 1b, H). However,
certain substituents were found to promote undesired intra-
molecular cyclizations, leading to peptoid truncations.10
Here, we introduce the use of hydrazones as submonomers
in solid phase peptoid synthesis that can afford both N-imino
and N-alkylamino glycine residues (Fig. 2), depending on the
conditions used to cleave the peptoids from the solid support.
Such monomers have shown promise in medicinal chemistry
platforms when incorporated into peptides, and exhibited
sufficient stability for biological testing.14 However, peptides
and peptidomimetics containing multiple different N-imino
glycines could not be accessed with previous methods, which
relied on the use of tert-butyl carbazate as a submonomer and
diversication of the N-amino glycine aer resin cleavage.14
Hydrazones are readily available via condensation of hydrazine
with aldehydes and ketones, providing easy access to a wide
array of side chain diversity. We demonstrate for the rst time
that they are readily incorporated during submonomer peptoid
synthesis, affording N-imino glycine- or N-alkylamino glycine-
containing peptoids following cleavage from the resin with tri-
uoroacetic acid (TFA) and various scavengers. We further
demonstrate that both N-imino glycines and N-alkylamino
glycines strongly induce trans-amide bonds, with introduction
Fig. 2 Structure and properties of (a) N-imino glycines and (b) N-
alkylamino glycines.
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Table 1 Optimization of peptoid cleavage from the resin
Entry
Cleavage cocktail
Time
Crude puritya (%)
6 : 7 ratio
1
2
3
4
5
6
7
8
9
10
TFA : H2O : TIPS 95 : 2.5 : 2.5
TFA : H2O : TIPS 95 : 2.5 : 2.5
TFA : H2O 95 : 5
TFA : H2O 95 : 5
TFA : TES 95 : 5
TFA : TES 95 : 5
TFA : TIPS 90 : 10
TFA : DCM: TIPS 45 : 50 : 5
TFA : DCM: TIPS 45 : 50 : 5
TFA : phenol 95: 5b
10 min
2 h
10 min
2 h
10 min
2 h
2 h
10 min
2 h
2 h
60
26
75
66
89
76
61
83
58
69
67 : 33
34 : 66
100 : 0
100 : 0
2 : 98
0 : 100
5 : 95
96 : 4
57 : 43
100 : 0
a
LCMS purity of the crude peptoid mixture at 214 nm (i.e., % area corresponding to the sum of 6 + 7). b The peptoid was precipitated in cold Et2O
before LCMS analysis.
We note that although TIPS efficiently reduced the
benzaldehyde-derived hydrazone side chain to give 7 aer 2 h
(Table 1, entry 7), using TES instead afforded a higher crude
purity overall with better selectivity for peptoid 7. Diluting
a 90 : 10 TFA : TIPS cleavage cocktail two-fold with dichloro-
methane yielded 96 : 4 and ꢀ1 : 1 mixtures of peptoid 6 and 7
aer 10 minutes and 2 hours, respectively (Table 1, entries 8 and
9). To prevent both hydrolysis and reduction of the N-imino
glycine residue, another cleavage solution using phenol as
a carbocation scavenger (95 : 5 TFA: phenol) was also tried,
which afforded 6 in comparable crude purities aer Et2O
precipitation as the 2 h 95 : 5 TFA: water cleavage (Table 1, entry
10). Overall, the excellent selectivity and good crude purities
observed using 95 : 5 TFA : H2O and 95 : 5 TFA : TES to give 6
and 7, respectively, encouraged us to pursue longer sequences
with more diverse N-imino glycine residues.
Increasing side chain diversity in N-imino- and N-alkylamino
glycine peptoids
To increase the diversity of side chains, we next evaluated the
synthesis and use of twelve different aliphatic and (hetero)
aromatic hydrazone submonomers. In the literature,
To study the utility of benzaldehyde hydrazone as a sub-
monomer in the synthesis of a longer oligomer, a 15mer sequence
was synthesized that contains ve N-imino glycine residues
separated by two N-(2-methoxyethyl)glycines (Nme) to increase
water solubility (Fig. 3a). Peptoid 8 was synthesized using an
automated peptide synthesizer, giving the desired product in 63%
crude purity aer resin cleavage with 95 : 5 TFA : H2O for 10
minutes. Gratifyingly, reduction of all ve N-imino glycine resi-
dues was also possible by using a 95 : 5 TFA : TES cleavage cocktail
for a total of 4 h (Fig. 3b), giving peptoid 9 as the major product.
Installing ve N-imino glycines in a row was also found to be
possible, albeit affording the desired peptoid in lower crude purity
(ESI†). In that case, the major side product appears to be a dele-
tion sequence lacking only one N-imino glycine residue.
Fig. 3 LCMS traces before purification at 214 nm of an oligomer
containing five N-imino glycine residues derived from benzaldehyde
hydrazone (a) cleaved using 95 : 5 TFA : H2O for 10 minutes (unre-
duced) and (b) cleaved with 95 : 5 TFA : TES for 2 h followed by
treatment of the crude peptoid with a fresh solution for another 2 h
(reduced).
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efficiency in solid phase submonomer peptoid synthesis. Here,
we chose to use N-(2-phenylethyl)glycine (Npe) as a spacer to
facilitate HPLC analyses of analogs containing N-imino glycines
with non-aromatic side chains. While installation of electron-
rich benzaldehyde hydrazones proceeded with no deletion
side product, the use of p-Br and p-CF3-benzaldehyde hydra-
zones as submonomers required optimization of the displace-
ment reaction, presumably due to the reduced nucleophilicity
imparted by the electron-withdrawing groups. Ultimately,
increasing the concentration of the hydrazone solution to 3 M
and placing the resin in a heated sonicator bath (60 ꢁC) for 2 h
provided pentamer 11d in good crude purity. Cleavage of this
peptoid from the resin using 95 : 5 TFA : TES provided reduced
12d in 74% crude purity, with an additional peak corresponding
to the parent peptoid with only one reduced hydrazone (11%).
Aer redissolving the crude peptoid in a fresh solution of 95 : 5
TFA : TES and stirring for an additional 2 h, full conversion to
pentamer 12d was detected in 84% crude purity. Displacement
with p-bromo benzaldehyde hydrazone was also performed in
a heated sonicator bath for improved yields.
Analogs 11e–g, containing heterocyclic moieties (e.g. pyri-
dine, pyrrole (unprotected), furan), were synthesized using
chloroacetic acid to avoid irreversible alkylation of the side
chain, as previously reported.19 When using these conditions for
the acylation step, addition of potassium iodide in the hydra-
zone submonomer solution was found to be necessary to give
the more reactive iodoacetamide aer in situ halogen exchange
(ESI†). With the exception of anilines, other amines are able to
displace the chloride,19 and the submonomer solution of
phenylethylamine was thus prepared without KI. From these
results, we established that hydrazones exhibit similar (low)
nucleophilicity compared to anilines. Yet, the high yield of most
hydrazone-containing peptoids using standard procedures
indicates that N-imino glycines are generally easier to install
than N-aryl glycines (e.g. peptoids 4a–g vs. 4h, vide supra). In the
process of this optimization, we began to wonder if an unde-
sired N-alkylation could also occur at the imino nitrogen when
using bromoacetic acid, lowering the overall crude purities in
all of our hydrazone-containing peptoids. However, when we
compared the two different monomer addition cycles with
several other sequences lacking heterocycles, we did not
observe an increase in yields using chloroacetic acid (data not
shown). This suggests that alkylation of the imino nitrogen by
bromoacetic acid is unlikely to be leading to signicant side
product formation.
Scheme 2 Hydrazone submonomer synthesis.
hydrazones are typically synthesized by reacting excess hydra-
zine monohydrate with an aldehyde in ethanol, and are used
immediately in their crude form aer an aqueous extraction
(Scheme 2).17 This simple one-step procedure should allow the
synthesis of hundreds of new peptoid submonomers from
common and oen inexpensive building blocks. Despite the
large excess of amine typically used in solid phase submonomer
peptoid synthesis (1–2 M solutions, 15–30-fold excess), we
found that the procedure to make the hydrazone submonomers
was critical for reproducible results. To access more water
soluble and volatile hydrazones (e.g. isobutyraldehyde hydra-
zone), we briey explored methods that bypassed aqueous
extractions.18 However, aer troubleshooting, the use of excess
hydrazine monohydrate followed by an aqueous work-up
proved essential for more consistent results in peptoid
synthesis (ESI†).§ Thus, all hydrazones in this study were ulti-
mately synthesized using this method (Scheme 2), and were
used immediately aerwards as freshly prepared solutions in
DMF.
With these different hydrazone submonomers in hand, we
synthesized pentamers 11–12a–l (Scheme 3) to test their
No reduction of the imine functional group in heterocyclic-
containing analogs 11e and 11f was observed using the 95 : 5
TFA : TES cleavage conditions over two hours. Conversely, N-
imino glycines with aliphatic side chains readily provided
reduced peptoids 12h-l under these conditions, and attempts to
obtain the parent compounds 11h-j in the absence of TES
Scheme 3 Crude purities of peptoid pentamers 11a–l and 12a–l at
a
214 nm. Hydrazone displacements were performed in a heated
sonicator bath. b Hydrazone displacements were performed with a 3 M
c
solution for 2 h in a heated sonicator bath. Crude purity after resulted in lower conversions due to rapid imine hydrolysis. It is
a second treatment with a fresh solution of 95 : 5 TFA : TES for 2 h. d
A
worth noting that in these cases we observed hydrolysis side
products even using a 95 : 5 TFA : phenol cleavage cocktail,
demonstrating that rapid hydrolysis could occur in the LCMS
vial with these analogs when dissolved in a MeCN : H2O
mixture. In comparison, peptoids with N-imino glycines
5 minute chloroacetylation was performed instead of a 25 min bro-
moacetylation, and the hydrazone displacement was performed in
a heated sonicator bath with a 1.5 M hydrazone solution made using
1 M KI in DMF. e The major side product corresponds to hydrolysis of
one or more hydrazone side chains.
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possessing cyclopentane (11k) and cyclohexane (11l) side chains enough for biological analysis at neutral pH.14 In the course of
were obtained as the major product when the LCMS analysis our studies, we discovered that acetylation of the N-terminus
was performed directly aer resin cleavage with 95 : 5 increased the stability of peptoids relative to their unacetylated
TFA : H2O for 10 min.
counterparts (ESI†). Moreover, dissolving the peptoids in 1 : 1
MeCN : H2O containing 0.1% triuoroacetic acid instead of
formic acid led to slightly faster hydrolysis and the appearance
of a side product over several days, likely arising from an acetyl
group transfer onto the N-amino glycine (ESI†). Hence, care
should be applied when purifying N-imino glycine-containing
peptoids by preparative HPLC containing 0.1% acid in the
mobile phase, and fractions should be immediately frozen and
lyophilized. Peptoids comprising of N-imino glycines with
aliphatic side chains can be cleaved from the resin with 95 : 5
TFA : TES v/v to access more stable (reduced) derivatives.
Hydrolytic stability of N-imino glycine-containing peptoids
Despite their improved stability relative to 11h–j, certain other
N-imino glycine-containing peptoids were found to undergo
hydrolysis to varying degrees over time. To get additional
insight about the relative stability of these peptoids, we moni-
tored select peptoid trimers (13a–d) in 0.1% formic acid (FA) in
1 : 1 MeCN : H2O for 8 hours and in phosphate buffer pH 7 for
several days (Fig. 4).{ These conditions were chosen to mimic
(1) the mobile phase that would be needed for reverse-phase
HPLC purications and (2) the mild aqueous conditions that
would be necessary to conduct biological assays. Aliphatic
hydrazones in analogs 13c–d underwent hydrolysis to a greater
extent under both conditions, with $50% hydrolysis observed
aer 4 days in phosphate buffer (pH 7). On the other hand,
aromatic hydrazones remained stable both in the presence of
0.1% FA over 8 h and in neutral aqueous buffer over days.
Moreover, addition of 1 mM Lys or Cys did not lead to or
increase decomposition rates for 13a–d (ESI†). Consistent with
the literature, we conclude that N-imino glycine-containing
peptoids possessing aromatic side chains should be stable
Synthesis of a peptoid with multiple different N-imino- and N-
alkylamino glycines
To further demonstrate the versatility of hydrazone sub-
monomers in solid phase peptoid synthesis, we synthesized
a peptoid possessing ve different N-imino glycines by auto-
mated solid phase peptoid synthesis (Fig. 5). All hydrazone
submonomers were freshly prepared as 1.5 M solutions in DMF
with 1 h displacement times, with the exception of 4-CF3-
benzaldehyde hydrazone (3 M, 4 h). The synthesis was paused at
the 7mer and 13mer stages, and resumed aer preparing fresh
solutions of the hydrazones needed to complete the 15mer.
Unfortunately, initial attempts to obtain peptoid 15 using 95%
Fig. 5 LCMS traces before purification at 214 nm of a peptoid 15mer
Fig. 4 Stability studies in (a) 0.1% TFA MeCN : H2O and (b) pH 7. % containing five different N-imino glycine residues, cleaved from solid
Hydrolysis was determined by integrating the LCMS peak areas for 13 support using (a) TFA : DCM: TIPS 45 : 50 : 5 for 10 minutes, and (b)
and 14 at 214 nm. The blue curve overlays with the gray curve and is 95 : 5 TFA : TES for 2 h, followed by a second treatment with a fresh
not visible.
solution of 95 : 5 TFA : TES for 2 h.
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TFA: 5% H2O during resin cleavage were unsuccessful. Upon
further investigation using hexapeptoids with two different N-
imino glycines as model compounds, we discovered that pep-
toids containing multiple different N-imino glycines could
undergo rapid hydrazone exchange under these conditions,
resulting in mixtures of products with scrambled N-imino
glycine compositions (ESI†). We hypothesized that a cleavage
cocktail that did not contain water would inhibit this undesired
hydrazone exchange. As previously demonstrated, 95%
TFA : 5% phenol could also be utilized to afford N-imino
glycine-containing peptoids (Table 1, entry 10). Ultimately, we
also found that exposing the resin to TFA : CH2Cl2 : TIPS
45 : 50 : 5 for 10 minutes afforded 15 in 52% crude purity
(Fig. 5a). Since TIPS is less efficient as a reducing agent
compared to TES, a shorter cleavage time coupled with a 2-fold
dilution of the cleavage cocktail with CH2Cl2 was able to provide
15 without reduction of N-imino glycines. Gratifyingly, hydra-
zone exchange was not observed upon dilution of the crude
peptoids with 0.1% FA in 1 : 1 H2O : MeCN or phosphate buffer
(pH 7). Cleavage from the resin with 95 : 5 TFA : TES, gave the
desired product (16) with all ve imines reduced in high crude
purity (Fig. 5b), conrming both the utility of hydrazone sub-
monomers in peptoid synthesis and the generality of the
TFA : TES cleavage conditions for reducing multiple different
hydrazones.
Conformational analysis of N-imino- and N-alkylamino
glycine monomers
Fig. 6 X-ray structures of 20 (left) and 21 (right).a A second structure
with opposite dihedral angles was present in the unit cell. b Disorder is
present in the piperidinyl region. In the minor conformation, j ¼ 150.6.
X-ray crystallography. While hydrazone submonomers offer
advantages in terms of side chain diversity and hydrogen
bonding capabilities, they also exhibit important structure-
inducing properties. Heteroatom substitution on the back-
bone nitrogen in both peptoids8–10 and peptides20 has been
shown to induce the trans-amide bond geometry, presumably adopted trans-amide bond congurations in the solid state in
due to electronic repulsion between the lone pairs of the side all cases (Fig. 6). Reduction of the C]N double bond in 20 to
chain heteroatom and backbone carbonyl group of the adjacent give 21 caused most notable changes in the side chain chi (c1)
residue when u ¼ 0ꢁ. To conrm this trend, model compounds dihedral angle, which pivoted from an average of 2.8ꢁ to ꢂ66.9ꢁ.
20a–c and 21a–c were synthesized in solution using similar In contrast, all backbone dihedral angles remained within 30ꢁ.
submonomer peptoid synthesis methods9 (Scheme 4), and their Moreover, for 21a–c, distances of 2.93–3.10 A between the side
˚
conformational properties were studied using NMR spectros- chain nitrogen (D) and carbonyl group (A) of the same residue
copy and X-ray crystallography.
with D–H/A angles of 112.4–122.0ꢁ revealed the presence of
Vapor diffusion of hexanes into ethyl acetate solutions of new intramolecular hydrogen bonds between the side chain NH
20a–c and 21a–c provided crystals of all compounds, which and the C-terminal amide carbonyl.21 In contrast, no hydrogen
bond was observed in N-acyl hydrazide monomers (Fig. 1b, type
H), where the side chain chi (c1) dihedral angle was closer to
90ꢁ.10a
NMR analysis. To study the conformational properties of N-
imino- and N-alkylamino glycines in solution, NMR analyses
were also conducted. Compound 23, which lacks a nitrogen
atom in the side chain but maintains the phenyl ring two atoms
away from the backbone nitrogen, was synthesized as a control
for conformational analysis in solution (Scheme 5). 1H NMR
analyses in CDCl3, CD3CN, and CD3OD revealed a single major
conformational isomer for 20a and two conformational isomers
for 21a and 23 (ESI†). NOESY spectra were acquired for 20a and
21a as representative examples, and provided evidence for
Scheme 4 Synthesis of peptoid monomers 20 and 21.
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trans 0.13–0.17). 1H NMRs collected over a range of temperatures
between ꢂ35 ꢁC and 45 ꢁC in CD3OD did not lead to the
appearance of a second peak in the acetyl region for 20a, or
cause the two peaks to coalesce in the case of 21a (ESI†). Control
peptoid 23 was also found to favor the trans isomer in all three
solvents, albeit to a lesser extent. When comparing Kcis/trans
values with other peptoid residues that favor the trans-amide
bond geometry, N-imino glycine derivative 20a appears to (1)
induce the trans-amide isomer more strongly than N-aryl glyci-
nes,2d,7a and (2) exhibit similar behavior to N-alkoxy glycines,
where no minor isomers are detected by 1H NMR.8,9 However, N-
alkylamino glycine derivative 21a is slightly less trans-amide
inducing compared to N-aryl glycines.
Scheme 5 Synthesis of control monomer 23.
In addition to favoring the trans-amide bond, reduced analog
21a has the potential for inter- and intramolecular hydrogen
bonding, implicating the side chain hydrogen-bond donor
(NH). To probe this unique hydrogen-bonding capability, we
monitored the NH chemical shi as a function of concentra-
Fig. 7 Observed NOE interactions in monomers 20a and 21a.
a trans-amide geometry in solution in both cases (Fig. 7), in tion, and conducted variable temperature (VT) experiments.
accordance with what was observed by X-ray crystallography. Little variation was observed in the NH chemical shi signal for
Specically, at 10 mM in CD3OD, NOE cross-peaks were present 21a in CDCl3 (Dd ¼ 0.01) over a concentration range of 1–
between the acetyl group (CH3) and the side chain phenyl ring 100 mM. This suggests that there is intramolecular hydrogen
for both 20a and 21a (ESI†), which is consistent with trans- bonding in solution,7b similar to what was observed in theꢁsolid
amide congurations.9 However, a weak cross-peak was also state (ESI, Fig. S26†). Moreover, VT NMR between 25–55 C in
observed between the acetyl group (Ha) and the backbone DMSO-d6 resulted in a chemical shi/temperature coefficient of
methylene protons (Hb) in both cases. While this is typically ꢂ0.59 ppb Kꢂ1, a value that is consistent with solvent shielding
unexpected for the trans-amide isomer,7a such weak NOEs have (ESI, Fig. S27†).22 Because most peptoid monomers lack
also been observed in at least one other trans-inducing peptoid hydrogen bond donors, these N-alkylamino glycine monomers
monomer.9 Compared to the NOE cross peak between the should exhibit unique properties that could be complementary
backbone methylene protons (Hb) and the piperidinyl protons to N-alkoxy glycines8 as hydrogen-bond acceptors. Notably, this
(Hc), which are cis to one another, integration of the acetyl/ trans-inducing monomer pair differ by a single nitrogen to
backbone methylene cross-peak was found to be 13-fold and oxygen atom substitution, which may promote productive side
8-fold less intense for 20a and 21a, respectively.k The weak NOE chain packing interactions.
observable between the acetyl CH3 and the backbone methylene
Computational studies. Theoretical calculations performed
is consistent with the atomic distance of 4.32–4.37 A between using the Gaussian 16 23 implementation of B3LYP24 density
the two protons detected by X-ray crystallography, which lies functional theory conrmed the trans-inducing capabilities of
˚
near the limit for NOE signal detection.
N-imino- and N-alkylamino glycine residues. The structures of
Kcis/trans values were determined for 20a, 21a and 23 in CDCl3, 20a and 21a were generated using the coordinates from their
CD3CN, and CD3OD (Table 2). While the presence of a minor respective X-ray crystal structures, and 23 by editing the crystal
conformational isomer was not detected by 1H NMR for N- structure of 20a in GaussView. Relaxed potential energy scans
imino glycine 20a (Kcis/trans < 0.05), in the case of 21a, the were performed about the u dihedral angle (Fig. 8). Energy
equilibrium favored the trans isomer in all three solvents (Kcis/ minima were optimized at the same level of theory but also
Table 2 Average Kcis/trans values in compounds 20a, 21a, and 23
1H-NMR (CDCl3)
1H-NMR (CD3CN)
1H-NMR (CD3OD)
DGcis/trans (kcal molꢂ1
)
Kcis/trans
DGcis/trans (kcal molꢂ1
)
Kcis/trans
DGcis/trans (kcal molꢂ1
)
a
a
a
Kcis/trans
20ab
21a
23
<0.05
—
<0.05
—
<0.05
—
0.13 ꢃ 0.01
1.22 ꢃ 0.06
0.17 ꢃ 0.01c
1.05 ꢃ 0.05
0.16 ꢃ 0.01
1.07ꢃ 0.05
0.26 ꢃ 0.05d
0.80 ꢃ 0.11
0.72 ꢃ 0.03d
0.19 ꢃ 0.02
0.44 ꢃ 0.01e
0.48 ꢃ 0.02
a
Average Kcis/trans and DGcis/trans values were calculated using values from 4 different concentrations in each solvent (ESI, Tables S11 and S12). b No
additional peaks for a minor conformer was detected for 20a in any solvent or concentration tested. c For peptoid 21a in CD3CN, the solvent peak
has the same ppm shi as the acetyl group; cis/trans ratios were measured using the backbone methylene peaks only. d Shoulder begins to appear
that may represent different rotamers, but are not fully resolved; cis/trans ratios were measured using the backbone methylene peaks only.
e
Shoulder begins to appear that may represent different rotamers, but are not fully resolved; cis/trans ratios were measured using the acetyl
peaks only.
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Fig. 8 Relative energy for relaxed potential energy scan of optimized
structures about the u dihedral angle for monomers 20a, 21a, and 23
reported at the B3LYP/6-31G(d,p)25 level of theory. See ESI† for more
computational details.
Fig. 9 Molecular and natural bonding orbitals (NBOs) for 20a. Orbitals
are shown at 0.045 isocontours.
lone pair on the N-imino nitrogen and the acetyl oxygen in 20a.
This can be observed in HOMO-1 (Fig. 9). NBOs 70 and 71 show
that these orbitals originate from a lone pair on the imino
nitrogen and acetyl oxygen respectively. When the nitrogen
atom is removed in 23, electron–electron repulsion is reduced
and there is only a small preference for the trans isomer. This
results in DGꢁcis=trans of 5.6 kcal molꢂ1 for 20a and 0.67 kcal molꢂ1
for 23 respectively. In 21a, the presence of a hydrogen bond
between the acetyl oxygen and the amino N–H bond (N–H/O ¼
included the D3 version of Grimme's empirical dispersion
correction.26 Energies were calculated at the same level of theory
with the 6-311+G(2d,p)27 basis set and included solvation
corrections in acetonitrile via the PCM method (Table 3).28
Consistent with experimental data, preference for the trans
isomer follows the order 20a > 21a > 23. The trends in free
energies, and consequently, the preference for the trans isomer,
can be explained by electron–electron repulsion between the
˚
2.24 A) reduces the energetic difference between the cis and
trans isomers. Thus, DGꢁcis=trans for 21a is smaller (2.9 kcal molꢂ1
)
Table 3 Calculateda energy differences between cis- and trans-
isomers
compared to 20a (5.6 kcal molꢂ1). Taken together, these results
support that N-imino glycines and N-alkylamino glycines have
a preference for the trans-amide conguration, which should
allow for secondary structure stabilization in longer oligomers.
Conclusions
In sum, we report here the use of hydrazones as new sub-
monomers for solid phase peptoid synthesis, providing easy
access to both N-imino- and N-alkylamino glycine-containing
peptoids. A wide array of aliphatic and (hetero)aromatic side
chains can be easily accessed using various hydrazone sub-
monomers, pre-synthesized from simple condensation reactions
with hydrazine and aldehydes. The N-imino glycine-containing
peptoids can be cleaved from the solid support using standard
TFA/H2O conditions when there is only one N-imino glycine or
when all the N-imino glycines in the sequence are the same, and
with TFA/TIPS/CH2Cl2 for 10 minutes or TFA/phenol when the
peptoid contains multiple different N-imino glycines. This is
essential to prevent undesired hydrazone exchange during resin
cleavage. Alternatively, resin-bound N-imino glycine-containing
peptoids can undergo a one-pot reduction/cleavage reaction
using TFA/TES to afford the N-alkylamino glycine-containing
peptoid. Both residues were found to exhibit a preference for
the trans-amide isomer, increasing the toolbox of structure-
inducing monomers for future peptoid design and
u Dihedral angle (ꢁ)
Molecule
cis
trans
DGcꢁis=trans ðkcal molꢂ1
Þ
20a
21a
23
ꢂ7.61
179
178
174
5.6
2.9
0.67
ꢂ4.94
ꢂ10.6
a
Energy calculations for each isomer were performed using B3LYP-D3/
6-311+G(2d,p)27 level of theory with the PCM solvation corrections
method (in acetonitrile) as implemented in Gaussian 16. A potential
energy surface scan for rotation around the C–N–N–H dihedral angle
was performed for the cis conformer of 21a, which identied a second
minima with hydrogen bonding to the acetyl carbonyl. See
computational methods in ESI for more details.
8408 | Chem. Sci., 2021, 12, 8401–8410
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applications. Moreover, the hydrogen-bonding capacity of N-
alkylamino glycines could be benecial in biological and mate-
rials applications, with potential to rigidify secondary structures
and intermolecular interactions.
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Conflicts of interest
There are no conicts to declare.
Acknowledgements
North Carolina State University is gratefully acknowledged for
startup support. All Nuclear Magnetic Resonance (NMR), High
Resolution Mass Spectrometry (HRMS), and X-ray experiments
were performed in the Molecular Education, Technology, and
Research Innovation Center (METRIC) at NC State University.
We thank Dr Roger D. Sommer for X-ray analysis of compounds
20a–c and 21a–c, Dr Peter Thompson and Dr Jennifer Sun for
assistance with NMR spectroscopy, and Danielle Lehman for
assistance with HRMS data collection. Funding for D8
VENTURE acquisition was provided in part by North Carolina
Biotechnology Center grant: NCBC#2019-IDG-1010. C. D. and
B. L. were supported by an NSF-funded (Integrated Computa-
tional and Experimental) (ICE) REU at North Carolina State
University (CHE-1659690) for part of this work. We thank Prof.
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computational studies and for overseeing the Chemistry REU
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