On-Resin Macrocyclization of Peptides Using Vinyl Sulfonamides as a Thiol-Michael "click" Acceptor

Article abstract of DOI:10.1021/acs.bioconjchem.8b00751

Macrocyclization of linear peptides imparts improved stability to enzymatic degradation and increases potency of function. Many successful macrocyclization of peptides both in solution and on-resin have been achieved but are limited in scope as they lack selectivity, require long reaction times, or necessitate heat. To overcome these drawbacks a robust and facile strategy was developed employing thiol-Michael click chemistry via an N-methyl vinyl sulfonamide. We demonstrate its balance of reactivity and high stability through FTIR model kinetic studies, reaching 88% conversion over 30 min, and NMR stability studies, revealing no apparent degradation over an 8 day period in basic conditions. Using a commercially available reagent, 2-chloroethane sulfonyl chloride, the cell adhesion peptide, RGDS, was functionalized and macrocyclized on-resin with a relative efficiency of over 95%. The simplistic nature of this process demonstrates the effectiveness of vinyl sulfonamides as a thiol-Michael click acceptor and its applicability to many other bioconjugation applications.

Full text of DOI:10.1021/acs.bioconjchem.8b00751

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Communication
On-Resin Macrocyclization of Peptides Using Vinyl
Sulfonamides as a Thiol-Michael ‘Click’ Acceptor
Bryan Sutherland, Bassil El-Zaatari, Nicole Irene Halaszynski,
Jonathan French, Shi Bai, and Christopher J Kloxin
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00751 • Publication Date (Web): 19 Nov 2018
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Bioconjugate Chemistry
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On-Resin Macrocyclization of Peptides Using Vinyl Sulfonamides as a
Thiol-Michael ‘Click’ Acceptor
Bryan P. Sutherland†, Bassil M. El-Zaatari‡, Nicole I. Halaszynski†, Jonathan M. French||, Shi Bai§, and
Christopher J. Kloxin*,†,‡
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†Department of Materials Science and Engineering, University of Delaware, 201 DuPont Hall, Newark, DE 19716, USA
‡Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716,
USA
||Department of Chemistry, Syracuse University, 111 College Place, Syracuse, NY 13210, USA
§Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA
ABSTRACT: Macrocyclization of linear peptides imparts improved stability to enzymatic degradation and increases potency of
function. Many successful macrocyclization of peptides both in solution and on-resin have been achieved but are limited in scope as
they lack selectivity, require long reaction times, or necessitate heat. To overcome these drawbacks a robust and facile strategy was
developed employing thiol-Michael click chemistry via an N-methyl vinyl sulfonamide. We demonstrate its balance of reactivity and
high stability through FTIR model kinetic studies, reaching 88% conversion over 30 min, and NMR stability studies, revealing no
apparent degradation over an 8 day period in basic conditions. Using a commercially available reagent, 2-chloroethane sulfonyl
chloride, the cell adhesion peptide, RGDS, was functionalized and macrocyclized on-resin with a relative efficiency of over 95%.
The simplistic nature of this process demonstrates the effectiveness of vinyl sulfonamides as a thiol-Michael click acceptor and its
applicability to many other bioconjugation applications.
Peptide conjugation is an ever-expanding field that allows for
the creation of complex structures1, molecular tagging2, and ma-
terial functionalization.3 Intramolecular conjugation reactions
within a peptide result in a cyclic structure, or macrocycle. Pep-
tide macrocycles are of great interest owing to their enhanced
resistance to protease degradation4 and improved potency.5 A
well-known example highlighting cyclic peptide advantages
over linear peptides is demonstrated in the simple cell adhesion
peptide sequence, RGDS.6 Higher stability and efficacy of cy-
clic peptides have led to further exploration into the synthesis
of macrocyclic motifs of various peptide sequences for biolog-
ical relevance7 or self-assembling structures.8
demonstrating click-like characteristics are particularly attrac-
tive for the synthesis of cyclic peptides, as they are selective,
proceed to full conversion, and produce little to no byprod-
ucts.22-23 One common click reaction used in peptide conjuga-
tion is the copper(I) catalyzed azide-alkyne cycloaddition (Cu-
AAC).24 The CuAAC reaction is a bio-orthogonal reaction be-
tween an azide and alkyne in the presence of copper(I) to form
a triazole product. For example, Ingale et al.15 demonstrated
on-resin macrocyclization of peptides using copper bromide
and excess sodium ascorbate; however, long reaction times of
18 h were required to reach sufficient conversions. Another
common click reaction used in peptide conjugation is the thiol-
ene reaction, which is the radical mediated addition reaction be-
tween a thiol and electron deficient alkene.25 For example, An-
seth and coworkers14 demonstrated a simple thiol-ene conjuga-
tion strategy utilizing cysteine as a thiol and a commercially
available allyoxycarbonyl (Alloc) protected lysine; however,
this approach required multiple additions of photoinitiator and
irradiation steps to perform the on-resin macrocyclization.
Macrocyclic peptides are often synthesized using solid-phase
peptide synthesis (SPPS) protocols and cyclizing the product
either in solution or on a solid substrate (i.e., on-resin). The so-
lution-phase macrocyclization route implements a conjugation
reaction on the fully protected peptide in dilute solution. Exam-
ples of solution-phase strategies include native chemical liga-
tion,9 imine formation,10 and palladium-catalyzed conjugation
reactions.11-12 However, solution-phase strategies largely neces-
sitate an additional deprotection step followed by purification
to remove cleaved protecting groups and oligomerized byprod-
ucts. The solid-phase macrocyclization route implements a con-
jugation reaction on the peptides while attached to the resin, re-
sulting in fewer side products and purification steps.13 The ad-
vantages of on-resin macrocyclization has made it the preferred
strategy for synthesizing cyclic peptide structure.
An attractive alternative click reaction to the CuAAC and
thiol-ene reactions is the thiol-Michael addition.26 In contrast to
the radical-mediated thiol-ene reaction, thiol-Michael additions
follow an anionic mechanism. Thiol-Michael reactions occur
between a thiol and an electron deficient alkene to form a stable
thioether bond in the presence of a base or nucleophile catalyst.
Thiol-Michael reactions have been exploited in a wide range of
biomolecular conjugation reaction, including peptides27, pro-
teins28, and nucleic acids.29 Common thiol-Michael acceptors,
such as maleimides and acrylates, exhibit fast kinetics and pro-
ceed to full conversion, but present complications when applied
to peptide chemistry.30-31 Maleimides are known to undergo a
ring opening reaction under aqueous conditions, both before
Several on-resin conjugation reactions have been employed
for the macrocyclization of peptides, including thiol-ene,14 az-
ide-alkyne cycloaddition,15-16 amide coupling17-18, glacial cou-
pling19, and ring-closing metathesis.20-21 In general, reactions
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and after conjugation,32-33 while acrylates are susceptible to hy-
drolytic cleavage, particularly after thiol addition.34 An alter-
native water stable thiol-Michael acceptor is the acrylamide
functional group; however, they exhibit slow kinetics and in-
complete conversions over long time periods.30 Here, we sug-
gest the vinyl sulfonamide as an efficient, water stable thiol-
Michael acceptor for peptide macrocyclization. Vinyl sulfona-
mides have been employed in both bioconjugations35 and poly-
mer networks36 for its high reactivity and hydrolytic stability.
Furthermore, simple installation of vinyl sulfonamides on pri-
mary or secondary amines is achieved using the commercially
available and inexpensive 2-chloroethanesulfonyl chloride.
1 eq of each of the alkenes, E2 (green, filled diamond), E3 (blue,
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5
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filled triangle), E4 (magenta, filled pentagon), E5 (red, filled cir-
cle), and E6 (orange, filled square) in the presence of DBU (1.4
mol%) in DMSO. (C) Similarly, 1 eq of the thiol, T2, was reacted
with 1 eq of each alkene, E2 (green, filled diamond), E3 (blue, filled
triangle), E4 (magenta, filled pentagon), E5 (red, filled circle), and
E6 (orange, filled square). E1 is not shown as it reached full con-
version with both T1 and T2 within the first 30 s of the experiment.
Solution-phase model reaction systems were performed to
compare relative rates of vinyl sulfonamides with several com-
monly implemented Michael acceptors (Figure 1A), such as
maleimide (E1) and acrylate (E2) as well as less reactive hydro-
lytically stable acrylamides (E3 and E4). The reaction kinetics
of thiol Michael conjugations are highly dependent on alkene
and thiol structure37. Model mono-vinyl sulfonamide com-
pounds, (E5) and (E6), were synthesized and compared with
maleimide, acrylate, and acrylamide functional groups. All
thiol-Michael acceptors were conjugated with two commer-
cially available thiols, n-butyl mercaptopropionate (T1) and a
cysteine analog (T2). Mercaptopropionic acid is readily conju-
gated to the N-terminus of a peptide or amine side-group on an
amino acid through amide coupling, whereas cysteine is a nat-
ural amino acid containing a thiol side-group.38
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The model maleimide and acrylate compounds proceeded
rapidly, while both acrylamides compounds showed minimal
conversion after 30 min under the same conditions. A summary
of the thiol-Michael kinetics is presented in Figure 1B and Fig-
ure 1C. The reaction of phenyl maleimide, E1, with T1 or T2
reacted to full conversion within the short time period of adding
and mixing them with 1,8-Diazabicyclo[5.4.0]undec-7-ene
(DBU) and transferring to the FTIR (30 seconds). Maleimide
functional groups showed enhanced reactivity for two distinct
reasons: alleviation of ring strain and the electron deficiency of
the olefin caused by the two adjacent activating carbonyl
groups.39 The kinetics of the methyl acrylate, E2, reacted with
either the alkyl or cysteine-based thiols proceeded rapidly,
reaching conversions of 92 ± 3% or 91 ± 1%, respectively, at 5
min. In comparison, reactions of either acrylamide functional
groups with either type of thiol exhibited sluggish kinetics. The
reaction of the secondary acrylamide, E3, with T1 or T2 reached
conversions of only 2 ± 1% or 1.5 ± 0.3% within 5 min, respec-
tively. Surprisingly, the reaction of the N-methyl tertiary acryla-
mide (denoted henceforth as a tertiary acrylamide), E4, with ei-
ther T1 or T2 had improved reactivity, reaching 7 ± 1% within
5 min. We attribute the poor reactivity of both acrylamides to
the decrease in electronegativity of the amides as compared
with esters.40 High electronegative moieties adjacent to olefins
result in a more reactive center for thiol-Michael conjugations.
Furthermore, the enolate that forms in amides is known to be
less stable as compared with ester-based groups.41-42 It is be-
lieved that an induction effect reduces the stability of the eno-
late species thereby resulting in slower kinetics of the secondary
acrylamide in comparison with the tertiary acrylamides.
Both secondary and tertiary vinyl sulfonamides exhibited
comparable kinetics to the acrylate, and much faster kinetics
than the secondary and tertiary acrylamides. The reaction of
secondary vinyl sulfonamide, E5, with either T1 or T2 reached
a conversion of 49 ± 1% or 21 ± 1%, respectively, over 5
minutes. As observed with secondary and tertiary vinyl acryla-
mides, the tertiary vinyl sulfonamide demonstrated faster kinet-
ics than the secondary vinyl sulfonamides. The reaction of ter-
tiary vinyl sulfonamide, (E6), reacted more rapidly than E5
Figure 1. Reaction kinetics of various thiols and electron poor al-
kenes. A) The general thiol-Michael reaction scheme, and the
model thiols and alkenes used in this study. B) Reaction kinetics
were monitored at room temperature over 30 minutes using FTIR
spectroscopy by tracking the disappearance of the thiol-peak be-
tween 2484 and 2545 cm-1. 1 eq of the thiol, T1, was reacted with
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Bioconjugate Chemistry
within the same timeframe with conversions reaching 76.4±
resin using a 95% TFA cleavage cocktail. This peptide was col-
lected, precipitated, and purified by HPLC to produce the linear
peptide with an overall yield of 10.0 mg or 14.2% based on the
initial resin’s molarity. The other two-thirds of the resin was
subjected to the macrocyclization procedure. A catalytic
amount of DBU in dimethylformamide (DMF) was added and
mixed for one hour. The resin was then washed multiple times
with DMF and DCM and was subjected to the resin cleavage
cocktail, precipitated, and purified by reversed-phase high-per-
formance liquid chromatography (RP-HPLC). The final prod-
uct was lyophilized resulting in 19.4 mg or 13.8% yield of fully
macrocyclized peptide. The structure of the fully cleaved mac-
rocyclized peptide is presented in Figure 3A. Based on the final
yield of the peptide the overall relative efficiency of the macro-
cyclization was >95%. Such a high efficiency of macrocycliza-
tion demonstrates the effectiveness of using vinyl sulfonamides
as a thiol-Michael acceptor for peptide macrocyclizations. All
attempts at cyclizing the peptide using a tertiary acrylamide
were unsuccessful at macrocyclizing on-resin further demon-
strating the superior reactivity of vinyl sulfonamides over
acrylamides. As expected, attempts at performing solution-
phase macrocyclizations under highly dilute conditions utiliz-
ing the same vinyl sulfonamide chemistry resulted in minimal
macrocycle product.13
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0.6% and 53.4± 0.7% with T1 and T2, respectively. The in-
creased reactivity of vinyl sulfonamides over vinyl acrylamides
and comparable reactivity to acrylates is attributed to two main
factors. First, sulfur atoms possess high polarizability leading to
a stronger inductive effect for stabilizing the α-carbanion that
forms during the thiol-Michael reaction mechanism.43 Second,
antibonding orbitals of sulfur atoms create σ-effects that further
stabilize the anionic center.44-46 The added stability of the α-car-
banion results in an increase in the kinetic performance for vinyl
sulfonamides in thiol-Michael conjugations. Additionally, it is
noteworthy that slower kinetics were experienced across all the
alkenes when reacting with the cysteine-based thiol T2. The
slower kinetics were an unexpected result based on the apparent
thiol pKa (between 6-9). However, the slower kinetics could be
attributed to neighboring groups providing steric hinderance or
affecting thiol pKa.
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Water stability of the thioether product from the thiol reaction
with either the secondary and tertiary vinyl sulfonamides was
evaluated and compared with the thioether product of an acry-
late. Sinha et al.36 showed that tertiary vinyl sulfonamide
demonstrated superior stability to acidic and basic environ-
ments in polymer networks as compared with networks formed
using secondary sulfonamides and acrylates. Using NMR spec-
troscopy, we confirmed the stability of both secondary- and ter-
tiary-vinyl sulfonamides in both acidic and basic media (Fig-
ures S16 to S21). Under basic conditions (in pH 10 borate
buffer), the model acrylate underwent rapid hydrolytic cleav-
age, while both vinyl sulfonamide compounds remained stable
over an 8 day period.
Owing to its rapid reaction kinetics and hydrolytic stability,
tertiary vinyl sulfonamide was selected to cyclize a ubiquitous
cell-adhesion peptide, RGDS; a process which has been shown
to both increase its stability to enzymatic degradation and effi-
cacy to promote cell spreading.6 The procedure for macrocy-
clizing the RGDS peptide is summarized in Figure 2. The pep-
tide sequence was preceded by a cysteine residue, bearing a
monomethoxy trityl (Mmt) protected thiol. The eventual re-
moval of the Mmt group through well-established protocols
provides a thiol chemical handle for the thiol-Michael addi-
tion.47 A D-phenylalanine residue was coupled after the cyste-
ine and prior to the rest of the sequence, which has been shown
to help promote macrocyclization of peptides.48-49 To install the
tertiary vinyl sulfonamide, a low-cost commercially available
Fmoc-protected sarcosine was added at the end of the peptide.
The Fmoc protecting group was removed revealing a secondary
amine to which the vinyl sulfonamide was added. To generate
the vinyl sulfonamide, a commercially available reagent, 2-
chlorosulfonyl chloride, was applied to the resin mixture in the
presence of triethylamine (TEA), yielding the Michael-acceptor
product. Macrocyclization of the peptide was catalyzed using
DBU, a common reagent used in Fmoc SPPS.50 Upon macrocy-
clization, a non-natural sulfonamide linkage is introduced into
the backbone of the macrocyclic peptide.
The macrocyclization of the peptide is achieved on-resin by
reacting the thiol of the cysteine with the tertiary vinyl sulfona-
mide end group. First, the resin was washed followed by the
selective deprotection of the Mmt protected cysteine, using a
dilute acid solution of 3% trifluoroacetic acid (TFA)/5% tri-
ethylsilane (TES) in dichloromethane (DCM). The peptide resin
was then neutralized using a dilute TEA solution and was col-
lected. One third of the resin was subjected to cleavage from the
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1
Figure 2. On-resin macrocyclization of an RGDS peptide. The fol-
lowing procedure was used to functionalize and macrocyclize the
peptide: (a) (4 eq) 2-chloroethanesulfonyl chloride, (8 eq) TEA, r.t.,
for 2 h, 2 times, (b) 3% TFA/5% TES in DCM, r.t., 30 s, 15 times,
and (c) 10 mol% DBU in DMF, r.t., 1 h.
disappeared post cyclization. Furthermore, the shifts in the H
spectra post-cyclization further signifying a structural change to
the peptide has occurred. For example, 1H chemical shifts in the
amide region (δ = 7.25 to 9.00 ppm) are broader prior to mac-
rocyclization. As linear peptides adopt a random coil confor-
mation, amide peaks form a broad distribution of conformations
demonstrated by the clustering of the protons between 8.00 and
8.25 ppm. Once a peptide forms a macrocycle, the discrete con-
formations are observed from the spreading of the amide pro-
tons as they adopt distinct orientations.
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1H NMR was performed to verify the disappearance of the
vinyl sulfonamide protons, denoted by ‘a’ and ‘b’ in Figure 3B.
As expected after undergoing cyclization on-resin, the vinyl
proton shifts (δ = 6.07, 6.78 ppm) disappeared from the cyclized
peptide spectrum. The thiol peak (δ = 2.00), denoted as ‘c,’ also
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Figure 3. Characterization of macrocyclic RGDS peptide. (A) Structure of the macrocyclized peptide cleaved from the resin with important
carbons for confirming macrocyclization numbered C1 through C4. The proton NMR (B) shows the linear peptide in blue and the cyclic
peptide in red. Alkenyl protons, denoted by ‘a’ and ‘b,’ disappear after macrocyclization. Additionally, the thiol peak ‘c’ disappears after
performing the macrocyclization. An overlay of 1H-13C HSQC (red/blue) and HMBC (grey) is shown in (C) with black arrows highlighting
1
the correlation between the newly formed thioether (C1) and cysteine residue (C3) for the macrocyclized peptide. For the edited H-13C
HSQC, the blue and red peaks correspond to CH2 or CH/CH3 carbons, respectively. The ESI-UPLC trace shown in (D) of the microcleaved
linear and macrocyclized peptides shows a shift in the elution time of the two peptide structures.
Correlation between protons using 2D NMR techniques fur-
ther confirmed the structure of the cyclic peptide. 1H-13C HSQC
and HMBC of the cyclic peptide were overlaid to compare the
single and multiple bond correlations between 1H and 13C peaks,
shown in Figure 3C. With these correlations, protons were as-
signed to the resulting thioether product and cysteine residue.
Upon macrocyclization, the thioether carbon (C3; δ = 34.08;
2.73/2.92 ppm) was used to determine the correlation with the
cysteine α-proton (C1; δ = 52.69; 4.39 ppm), as denoted by the
black arrows. These correlations confirm that the thiol-Michael
reaction has occurred. Furthermore, COSY, NOSEY, and
TOCSY NMR data were collected on both the linear and mac-
rocyclized peptides to confirm these findings (Figure S28 to
S43).
On-resin microcleavages were performed on the linear and
macrocyclic peptides for further confirmation of macrocycliza-
tion. A shift was observed when analyzing the macrocyclized
peptide which eluted at 2.69 min, while the linear peptide eluted
at 2.79 min. Upon macrocyclization, the structural changes in
the peptide results in a faster elution time on the UPLC column.
The R groups of the amino acid residues are forced outward al-
lowing for more direct interactions of the polar R groups with
the hydrophobic C18 column packing resulting in a faster elu-
tion time. A shift in retention time was again observed during
purification of the linear and macrocyclized peptide using RP-
HPLC (Figures S2 and S3).
A facile protocol for macrocyclization of peptides using ter-
tiary vinyl sulfonamides was established and shown to be com-
patible with standard Fmoc-based SPPS protecting groups and
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Bioconjugate Chemistry
7.
Bokesch, H. R.; Pannell, L. K.; Cochran, P. K.; Sowder, R.
methods. This functional group is easily generated using any
secondary amine handle utilizing a low-cost commercially
available reagent, 2-chlorosulfonyl chloride. Additionally, it
was shown to possess comparable reactivity to acrylates and
was considerably more reactive than acrylamides. This func-
tional handle is stable in harsh basic environments unlike acry-
lates. The higher stability of tertiary vinyl sulfonamides sug-
gests that it can be applied in applications where the conjugation
bond stability is desired. Due to the wide availability, low-cost,
high selectivity, and increased stability of the tertiary vinyl sul-
fonamide, we expect the scope of this thiol-Michael click reac-
tion to expand beyond peptide macrocyclization and broaden
the toolbox of reaction handles for thiol-Michael conjugations.
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Funding Sources
This work was supported by DOE grant DE-SC0019355. This
project was partially supported by the Delaware COBRE program,
with a grant from the NIH NIGMS (5 P30 GM110758-02).
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Author Contributions
All authors have given approval to the final version of the manu-
script.
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Asfaw, H.; Wetzlar, T.; Martinez-Martinez, M. S.; Imming,
Notes
The Authors declare no competing financial interest.
19.
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E., Rigid Peptide Macrocycles from On-Resin Glaser Stapling.
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Secondary Structure through Ring-Closing Alkyne Metathesis.
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