One-Pot Cyclization and Cleavage of Peptides with N-Terminal Cysteine via the N,S-Acyl Shift of the N-2-[Thioethyl]glycine Residue

Magdalena Wierzbicka, Mateusz Waliczek, Anna Dziadecka, and Piotr Stefanowicz*

Note

One-Pot Cyclization and Cleavage of Peptides with N‑Terminal

Cysteine via the N,S -Acyl Shift of the N ‑2-[Thioethyl]glycine Residue

Cite This: https://doi.org/10.1021/acs.joc.1c01045

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sı Supporting Information

ABSTRACT: We developed a one-pot method for peptide

cleavage from a solid support via the N,S-acyl shift of N-2-

[

thioethyl]glycine and transthioesteriﬁcation using external thiols

to produce cyclic peptides through native chemical self-ligation

with the N-terminal cysteine. The feasibility of this methodology is

validated by the syntheses of model short peptides, including a tetrapeptide, the bicyclic sunﬂower trypsin inhibitor SFTI-1, and

rhesus Θ-defensin RTD-1. Synthesis of the whole peptide precursor can be fully automated and proceeds without epimerization or

dimerization.

yclic peptides are an important class of biologically active

natural products. Their conformational stability enables

combining the advantages of both NCL and solid-phase

,11

peptide synthesis (SPPS).

Recently, Dawson’s o-amino-

selective binding to their receptors. The biological signiﬁcance

makes them interesting targets for drug development, which in

turn necessitates the syntheses of their multiple variants. The

cyclization of linear precursors is the yield-determining step

(methyl)aniline (MeDBz) linker was applied for the on-resin

25,26

one-pot preparation of Kalata B1 and McoTI-II, SFTI-1,

and cyclotetrapeptides. Another example of this approach is

N-ethylcysteine-mediated ligation. However, this method is

−3

and can be reached in several ways.

extensively used methodologies is native chemical ligation

One of the most

limited by the less-eﬃcient N-acylation of N-ethylcysteine,

which necessitates the coupling of a preformed in-solution

dipeptide to the solid support. In this article, we employed the

novel building block N-2-[thioethyl]glycine that can be

synthesized from commercially available substrates, loaded

on the solid support by a submonomeric approach, and is

susceptible to amide−thioester rearrangement, allowing intra-

molecular native chemical ligation with the N-terminal cysteine

(Scheme 1). The simplicity of the synthesis of the peptide

precursor, which can be automated, and the one-pot

cyclization were the main advantages of the proposed methods

over described protocols.

(

NCL) developed by Kent et al., which is based on the

chemoselective reaction between peptide segments containing

N-terminal cysteine and C-terminal thioester groups. Peptide

thioesters with a N-terminal cysteine undergo intramolecular

NCL to yield homodetic cyclic peptides. However, the

synthesis of peptide thioesters by Fmoc-based solid-phase

peptide synthesis is challenging due to the instability of

thioesters under the piperidine-mediated Fmoc deprotection

step and requires the so-called safety-catch linkers, for example,

−8

sulfonamide,

linkers.

trithioortho esters, or aryl and hydrazine

0,11

The peptide thioesters also can be formed by N,S-

acyl shift and transthioesteriﬁcation with external thiols.

Selenocysteine and N- and α-alkyl cysteines are widely

Scheme 1. Mechanism of the One-Pot Cyclization

2,13

known as thioester precursors and surrogates.

Additionally,

14,15

bis(2-sulfanylethyl)amido (SEA),

N-sulfanylethylanilides

(

SEAlides), and thioethylalkylamido (TEA) surrogates are

18,19

commonly used.

They were applied in the synthesis of

20 21

cyclic peptides, including SFTI-1, cyclopsychotride, con-

otoxin MVII, kalata 1, and McoTI-II. One of the recent

additions to the above-mentioned class of thioesteriﬁcation

devices is the N-(2-hydroxybenzyl)cysteine, which is inspired

by the natural intein splicing process. The cyclization in native

conditions is the method of choice for the preparation of

bicyclic and cysteine-rich peptides due to the spontaneous

oxidative formation of disulﬁde bridges. Currently, some on-

resin peptide NCL cyclization methods are being developed.

This approach reduces the number of synthetic steps and

simpliﬁes peptide puriﬁcation from excess reagents, thus

Received: May 4, 2021

XXXX The Authors. Published by

American Chemical Society

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

cyclic peptides containing cysteine residues, (Figure 1).

Note

Our methodology is validated by the synthesis of diverse

target peptide precursors (Scheme 2b) were then assembled

on the solid support either manually via a sonication-assisted

Fmoc-SPPS protocol or automatically. The solid-phase

synthesis of peptide precursors was monitored by BrCN

cleavage, followed by LC-UV-MS. We compared several

conditions for the manual, partially automated, and fully

automated (with or without microwave heating) syntheses of

peptide precursors with diﬀerent durations for (trt)cysteamine

incorporation (Figure S6 ). We observed that the reaction of

(

trt)cysteamine with the solid support can be completed in 1−

h instead of overnight when using microwave-assisted

heating at 75 °C. The microwave heating should not be

applied for the coupling of chloroacetic acid.

After the Fmoc-SPPS of the peptide precursors, we tested

multiple conditions to study the rates of the N,S-acyl shift,

transthioesteriﬁcation, and intramolecular ligation in neutral

and acidic conditions in the presence of various thiol additives.

They are listed in Table 2 in the Peptide Cyclization

Table 1. Characterization of Synthesized Peptides

Figure 1. Structures of cyclic peptides 1−5 obtained by the presented

method. The N-terminal cysteine moiety is marked in green.

TentaGel-NH resin, which is compatible with both organic

and aqueous reaction conditions, was chosen. For the ﬁnal

deprotection that maintained the peptide precursor on a solid

support, we used an orthogonal AAM linker that could be

cleaved by BrCN. For longer polypeptides, we replaced alanine

residues with either β-alanines or polyethylene glycol to

increase the distance between the solid support and the

peptide chain. They were tested on the model sequence

CAKPGG-N-2-[thioethyl]glycine, and we found that the type

of linker had no impact on the product formation (SI 4). For

the synthesis of our device, we used commercially available 2-

Procedures section and are described in detail. The peptidyl

resin was incubated in the buﬀer containing 50 equiv of an

external thiol in the presence of a reducing agent at 40−50 °C

for 48 h to promote the N,S-acyl shift and transthioester-

iﬁcation, leading to the release of the peptide-active thioester

from a solid support. The ﬁnal step involves the intramolecular

NCL reaction between the N-terminal cysteine residue and the

C-terminal thioester, which aﬀords the desired head-to-tail

cyclic product. Cyclization was initially performed with

MESNa in citrate buﬀer, which was then followed by a second

transthioesteriﬁcation with MPAA in phosphate buﬀer to

enhance the rate of NCL (procedure A). These reaction

(

thio)ethamine (cysteamine) hydrochloride and performed the

thiol protection using both trityl (trt) and 4-methoxytrityl

mmt) chlorides to synthesize (trt)cysteamine 6 (Scheme 2a)

(

Scheme 2. Synthesis of S-Trityl Cysteamine 6, (a) Its

Incorporation into the Solid Support, and (b) Further

Peptide Precursor Synthesis and Deprotection

conditions were adopted from Taichi and were subsequently

shortened to a one-pot procedure with either MESNa or

MPAA as external thiols (procedures B and C, respectively).

We employed the thioesteriﬁcation method with MESNa

(

procedure B) for short peptides as it was easier to remove its

excess by SPE. Furthermore, the incubation of the peptide

precursor in phosphate buﬀer containing MPAA and TCEP for

8 h leads to the desulphurization of the cyclized product.

However, procedure B has not worked for the long precursors

of 4 and 5; therefore, we switched to the more reactive MPAA

(

procedure C) and replaced phosphate with TEAB (triethy-

and (mmt)cysteamine (6a, SI 1.2). Their syntheses were

lammonium bicarbonate) buﬀer. In procedure B1 we did not

add any thiol. Additionally, the one-pot peptide cyclization was

performed in solution (procedure D). The best results were

obtained for procedures A−C, which were applied for the

syntheses of target peptides 1−5, and the corresponding

cyclization yields are listed in Table 1. The obtained peptides

were desalted, puriﬁed by preparative RP-HPLC, and

previously reported by O’Neil and Riddoch, but herein we

adapted the procedure from Barlos, proceeding in mild DMF

conditions. To insert the S-protected cysteamine, the resin that

was preloaded with the AAM sequence was coupled with

chloroacetic acid (Scheme 2a). Then, the S-protected

cysteamine was introduced through S 2 substitution. The

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

bonds. The LC-MS data of RTD-1 are presented in Figure 2 .

Note

subsequently analyzed by LC-MS. The cyclization yields were

cleavage in two sequences: AAAAA, which has no N-terminal

cysteine, and CPKA. This experiment demonstrates that our

device works well for peptide cyclization even without an

additional thiol, although it is less eﬃ cient for thioester

formation in heterogeneous conditions (Figure S18).

calculated by comparing the amount of the puriﬁed peptide to

the precursor substitution level determined spectroscopically.

The overall yields were calculated, corresponding to the initial

resin loadings.

First, we performed the syntheses of model cyclic peptides

with 4−7 amino acid residues. When studying the cyclization

conditions for the model peptide CFGPKA, we observed that

not only the intermediate H−CFGPKA−MESNa (1a) was

produced after the precursor treatment with MESNa in citrate

buﬀer at pH 3 overnight but also the ﬁnal cyclized product (1).

This proved that in the case of peptide precursors with N-

terminal cysteine, the cyclization is a one-pot process. The

isolated thioester dissolved in water underwent self-ligation to

Switching to longer bioactive peptides, we synthesized the

sunﬂower trypsin inhibitor SFTI-1 (4, Figure 1). This

bicyclic peptide contains two cysteine residues and can be

synthesized using two diﬀerent N-terminal cysteine peptide

precursors. We chose the precursor sequence CTKSIPPICFP-

DGR-N-2-[thioethyl]glycine-linker to avoid the less-reactive

C-terminal Ile in a sequence. Additionally, the proline residues

located in the middle of the chain facilitate the β-turn

formation. The puriﬁed monocyclic 4 was then subjected to air

oxidation for 48 h to form the disulﬁde bridge. The natively

oxidized product is predominant; however, we also observed

traces of intermolecular disulﬁde bond formation (Figures

S41 −S42). In addition, we also synthesized the SFTI-1

precursor using ChemMatrix resin with a Rink linker. This

allowed for the TFA-based cleavage of the whole construct,

including the linker. Then, we performed the cyclization in

solution according to the aforementioned procedure D. This

experiment, in turn, enabled an insight into the degree of

substrate conversion. LC-MS analysis revealed relatively high

conversion (80%) of the substrate toward the cyclic product

(Figure S45). The identity of the obtained bicyclic SFTI-1 was

conﬁrmed by a direct LC-MS comparison with a reference

and hydrolysis to linear H−CFGPKA−OH 1b (Figure S24),

while when incubated in phosphate buﬀer (pH 7.4) with DTT

at 40 °C it underwent a complete transformation into 1

(Figure S25). Eventually, we established procedure B for the

full cyclization of 1, where the peptidyl resin was ﬁrst

incubated in citrate buﬀer with 50 equiv of MESNA at 40

°C for 24 h. Then, 10 equiv of DTT in phosphate buﬀer was

added, and the mixture was incubated in the same conditions,

which provided the cyclization yield of 26%. The correspond-

ing LC-UV-MS data are presented in SI 6.1 . The next

investigated peptide was the cysteine analog of axinellin A (2),

a cytotoxic peptide isolated from marine sponges (Figure 1).

Here we report the data for the synthesis with MPAA in the

TEAB buﬀer medium (procedure C) that yielded 55% of 2.

The ﬁnal workup involved the acidiﬁcation with TFA and an

extraction with diethyl ether to remove the excess MPAA and

DTT. Moreover, because of the usage of a volatile buﬀer, there

was no need for additional desalting, and only lyophilization

was suﬃcient.

sample provided by Rolka, and both LC-MS and MS spectra

gave identical fragmentation patterns (Figure S44).

Following the presented approach, we also synthesized the

rhesus Θ-defensin RTD-1 (5, Figure 1), which is a cysteine-

rich antimicrobial peptide that plays a signiﬁcant role in the

mammalian innate immune system. We chose to form a Phe−

Cys peptide bond through NCL and therefore located the Phe

residue on the C-terminus of the linear precursor (CRCLC-

RRGVCRCICTRGF-N-2-[thioethyl]glycine-linker). It was

proven that ligations at the C-terminal Phe proceed

Native chemical ligation is considered a racemization-free

process. To evaluate epimerization in the N-2-[thioethyl]-

glycine system, we have synthesized [D-Thr ,Cys ]axinellin A

2a) and compared its retention time in LC-MS with that of its

(

L-Thr isomer. This conﬁguration change altered the retention

time by more than 1 min, allowing the detection of the

epimerized product, which in our case was below 1% (Figure

S30).

eﬃciently. The cyclization yield of peptide 5 was almost

40%. The last step involved oxidation to form three disulﬁde

The cyclic hexa- and heptapeptides were relatively easy to

synthesize; however, the formation of the cyclic tetrapeptide

was accompanied by the formation of the cyclic octapeptide. In

the case of CPKA, a large volume of solvent in the second

reaction step (10 mL for 5 mg of peptidyl resin) allowed the

formation of cycloCPKA 3, while a lower volume (2 mL)

favored the cyclodimerization toward the cyclic octapeptide

with a doubled sequence, cycloCPKACPKA 3a. The

separation and analysis of the cyclic monomer and dimer

were challenging since both peptides underwent fast oxidation,

producing inter- and intramolecular disulﬁde bonds. 3a and

oxidized 3 are structural isomers with the same m/z value and

similar retention times (rts); thus, during puriﬁcation and

analysis, a 10 mM TCEP solution was added to maintain the

reduced form. Therefore, in section SI 6.4.1 we present the

analytical data for both forms. Both peptides were obtained

with yields of around 20%. To exclude the possible formation

of a cyclodimer, we tested the self-ligation without any

additional thiol. This approach gave the ﬁnal product 3 (Figure

S36 ), thus conﬁrming the direct on-resin self-ligation. This

result brought us to reconsider the role of an external thiol in

cyclization. Therefore, we compared the susceptibility for

Figure 2. Analytical data obtained for puriﬁed and oxidized Θ-

defensin RTD-1 5. (A) LC-ESI-MS spectrum. (B) MS spectrum with

the expanded isotopic pattern of [M + 4H] : m/z 520.9864. (C) LC-

MS chromatogram (total ion current, TIC), LC-ESI-qTOF-MS

instrument. (D) HPLC chromatogram at 210 nm, rt = 4.77 min,

LC-UV-ESI-IT-TOF instrument.

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Note

An intramolecular disulﬁde bond arrangement for the oxidized

defensin has not been tested, however, literature data clearly

show that the oxidation of the cyclic precursor of Θ-defensin

results in the formation of the native conﬁguration of disulﬁde

bridges. We applied the oxidation method of Conibear that

provided a one sharp chromatographic peak corresponding to

the molecular mass of oxidized Θ-defensin. Therefore, we

assumed that the structure of the obtained product was

identical to that of the native one.

MS/MS. The LC-UV-IT-TOF instrument is a hybrid system

consisting of a liquid chromatograph, a PDA detector, an ion trap,

and a time-of-ﬂight mass analyzer. In both instruments, CID

fragmentation (with Argon) was used, and the potential between

the spray needle and the oriﬁce was set to 4.5 kV. The LC systems

were operated with the following mobile phases: A = 0.1% HCOOH

in H O and B = 0.1% HCOOH in MeCN in a gradient separation

from 0 to 60% B/A in 15 min at a 0.2 mL/min ﬂow rate and a 2 μL

injection. For highly hydrophilic peptides, the isocratic conditions

were applied. The separations were performed on an Aeris Peptide

XB-C18 column (100 mm × 2.1 mm, 1.7 μm bead diameter). If

mentioned, the column was thermostated. More hydrophobic peptide

samples (4, 5, and their derivatives) were dissolved in 400 μL of a

To conclude, we have demonstrated that the N-2-

thioethyl]glycine moiety, which undergoes a N,S-acyl shift,

[

can be applied for the synthesis of cyclic peptides containing

cysteine residues. Through the one-pot cyclization, the yield of

the synthesis can be improved as it avoids the isolation and

puriﬁcation of unstable thioesters. We also conﬁrmed that the

proposed method is essentially epimerization-free. In addition,

the synthesis of N-2-[thioethyl]glycine is straightforward and

can be easily assembled directly on a polymeric support using

inexpensive and commercially available reagents. Our linker

can be further eﬃciently derivatized directly on the resin both

manually and automatically. Presented studies suggest that

both the following mechanisms take place: in the ﬁrst step, the

peptide thioester is released from the solid support as a result

of the N,S-acyl transfer and transthioesteriﬁcation, which next

undergoes the intramolecular NCL reaction to result in the

formation of a homodetic cyclic peptide. The second

mechanism proceeds by direct N-terminal cysteine self-ligation

without an external thiol. Our methodology simpliﬁes the

synthesis of biologically relevant cyclic peptides over protocols

described in the literature.

water/acetonitrile mixture (95:5). H NMR and C{ H} NMR

spectra were recorded on a high ﬁeld Bruker 500 MHz spectrometer

equipped with a broadband inverse gradient probe head. Spectra were

referenced to the residual solvent signal (CDCl at 7.24 ppm or

MeOD at 4.87 ppm). The deuterated solvents were purchased from

Sigma-Aldrich. For the measurements of the substitution level, a UV−

vis Plate reader Tecan inﬁnite M200 Pro instrument was used (Tecan

Group Ltd., Mannedorf, Switzerland) in the cuvette measurement

mode with blanking in the range of 230−700 nm.

Reagents and Materials. All commercially available reagents

were used without further puriﬁcation, and water was deionized by

the reverse osmosis system (Hydrolab, Poland). Solvents for building

block synthesis and peptide synthesis are as follows: chloroform

(

stabilized with amylene), CDCl ; dimethylformamide, DMF;

dichloromethane, DCM; methanol, MeOH; tetrahydrofuran, THF;

diethyl ether, Et O; N,N-diisopropylethylamine, DIPEA; piperidine,

PIP; HCOOH; triﬂuoroacetic acid, TFA; cyanogen bromide, BrCN;

triisopropylsilane, TIS; and 1,2-ethanedithiol, EDT. Solvents in the

analytical grade were obtained from Sigma-Aldrich, and acetic

anhydride was obtained from Lachner. Fmoc-amino acid derivatives

for peptide synthesis were purchased from PeptideWeb, except for

Fmoc−Cys(Mmt)−OH and Fmoc−Lys(Mtt)−OH (Sigma-Aldrich).

Coupling reagents are as follows: TBTU, O-(benzotriazole-1-yl)-

N,N,N′,N′-tetramethyluronium tetraﬂuoroborate; PyBop, benzotria-

zole-1-yl-oxytripyrrolidinophosphonium hexaﬂuorophosphate; and

PyAOP, 7-azabenzotriazol-1-yloxytrispyrrolidinophosphonium hexa-

ﬂuorophosphate. Reagents were purchased from Navoabiochem,

N,N′-diisopropylcarbodiimide (DIC) was purchased from Fluka and

Oxyma Pure was purchased from Iris Biotech. The resins for SPPS are

as follows: H-Rink amide ChemMatrix resin (0.40−0.60 mmol/g)

was purchased from Sigma-Aldrich, and TentaGel HL NH2 (0.56

mmol/g and 0.26 mmol/g) and TentaGel MB NH2 (0.23 mmol/g)

resins were purchased from RAPP Polymers GmbH. For the N-2-

[thioethyl]glycine synthesis, cysteamine chloride, 4-methoxytrityl

chloride (mmt-Cl), trityl chloride (trt-Cl), chloroacetic acid, and

bromoacetic acid were purchased from Sigma-Aldrich. For peptide

cyclization, sodium 2-mercaptoethanesulfonate, MESNa, 4-mercapto-

phenylacetic acid, MPAA, tris(2-carboxyethyl)phosphine, TCEP,

dithiothreitol, DTT, triethylammonium bicarbonate buﬀer, and

TEAB (1M, pH 8.5) were purchased from Sigma-Aldrich. Other

chemicals, including citric acid (monohydrate), hydrated disodium

phosphate, POCH, hydrated monosodium phosphate, were purchased

from AppliChem, NaOH was purchased from Stanlab, and analytical

grade HCl was purchased from Sigma-Aldrich. Solvents for LC-MS

are as follows: MeCN and HCOOH in HPLC grade were purchased

EXPERIMENTAL SECTION

General. Solid-phase peptide synthesis according to the FMOC

strategy (Fmoc SPPS) was performed manually with an ultrasound-

assisted coupling method developed in our research group in

syringe reactors equipped with ﬁlters (Intavis). Automated peptide

synthesis was performed on a Microwave Peptide Synthesizer Initiator

■

Alstra (Biotage, Sweden) instrument. For desalting and solid-phase

extraction, either Sep-Pak C18 Plus short cartridges (360 mg sorbent,

5−105 μm particle size, 125 Å pore size, pH 2−8, Waters) or OMIX

C18 pipet tips for microextraction (10−100 μL, Varian) for

microscale monitoring were used. The synthesized peptides were

puriﬁed using preparative HPLC on a Varian ProStar (Palo Alto, CA)

spectrometer equipped with the UV detector at 210 and 280 nm, with

the column TSKgel ODS-120T (215 × 30 mm, 150 Å, 10 μm), a ﬂow

rate of−7 mL/min, and the following eluents: A = 0.1% TFA in H O

and B = 0.1% TFA in 80% MeCN/H O, with a gradient of 0−40% B/

A in 40 min. For peptide characterization, an analytical Thermo

Separation HPLC system with UV detection (210 nm) was used with

a Vydac Protein RP C18 column (4.6 × 250 mm, 5 μm) and a

gradient elution of 0%−80% B/A in 40 min (A = 0.1% TFA/H O and

B = 0.1% TFA in 80% MeCN/H O, ﬂow rate of 1 mL/min) as well as

HRMS via an ESI-FT-ICR Apex-Qe 7T instrument (Bruker) in the

positive ion mode. For fragmentation, the collision-induced

dissociation (CID) technique was used with argon as the collision

gas. The potential between the spray needle and the oriﬁce was set to

from Sigma-Aldrich, and MeOH and HPLC grade H

were purchased from J. T. Baker.

O in HPLC

.5 kV. The MS was calibrated with a Tunemix mixture (Bruker

Daltonics) following a quadratic method. Samples were dissolved in

.1% HCOOH in 50% MeCN/H O. Additionally, for 6 and 6a, a

Synthesis of Protected Cysteamines 6 and 6a. Both S-

protected cysteamines with trityl and 4-methoxytrityl groups were

synthesized according to the procedure adopted from Barlos et al. as

described for 4-methoxytrityl-S-cysteine in a mild DMF medium.

Synthesis of 2-[(Triphenylmethyl)sulfanyl]ethan-1-amine 6.

Cysteamine hydrochloride (5.68 g 50 mmol) was dissolved in 50

mL of DMF in an Erlenmeyer ﬂask equipped with a magnetic stirrer ,

and to the ﬂask was added 13.94 g (50 mmol) of trityl hydrochloride.

The mixture was stirred at room temperature for 3 h. The resulting

solution was concentrated under the stream of nitrogen, then

−

NaCl solution was added to the ﬁnal concentration of 10 mmol.

LC-MS experiments were performed on Shimadzu LCMS-9030 and

Shimadzu LC-UV-IT-TOF instruments in the positive ion mode with

electrospray ionization. The LCMS-9030 instrument was equipped

with a UHPLC Nexera X2 system and a hybrid mass analyzer, which

was a single quadrupole coupled with time-of-ﬂight mass analyzer

(

qTOF) at the m/z range of 100−1500 and a range of 100−2000 for

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Note

neutralized with KOH/MeOH to a pH of 9 and evaporated overnight

under the stream of nitrogen. The resulting mixture was extracted four

times with DCM (4 × 50 mL), then all fractions were collected,

washed with water (2 × 25 mL) and brine (2 × 25 mL), dried over

glycineAA-HL] : 3.6−3.8 min, m/z [M] Calcd for C H N O S

28 48

10 2

360.1462, found 360.1450 (HL = homoserine lactone).

Manual Peptide Precursor Synthesis on Peptidyl Resin and

Its Activation. The syntheses of peptide precursors were performed

on the N-2-[(trt)thioethyl]glycine-AAM-resins or N-2-[(trt)-

thioethyl]glycine-βAβAM-resins described above according to the

same standard Fmoc strategy. The only exception was the coupling of

the next Fmoc-protected amino acid to the N-2-[(trt)thioethyl]-

glycine residue. In this case, 6 equiv of the Fmoc-protected amino

acid was added twice with 6 equiv of PyAOP and 12 equiv of DIPEA.

After the coupling of the last amino acid residue (cysteine), the Fmoc

was not removed, and the peptidyl resin was dried and placed in the

desiccator. Then, 5 mg of the resin with the peptide precursor was

swelled in 1 mL of 20% PIP/DMF for 20 min to determine the

substitution level (see below Fmoc Substitution Level Determina-

tion). Next, the dried peptidyl resin was subjected to the ﬁnal

deprotection and cleavage with 20 μL of 3 M BrCN/DCM plus 200

MgSO , and evaporated on a rotary evaporator. The ﬁnal product was

crystallized from EtOAc by adding small portions of hexane. Crystals

were left for growth overnight at 4 °C; yield: 8.07 g (25.27 mmol

−

50.54%) of a yellowish powder. TLC: 5% MeOH/CHCl , rf = 0.29,

mp = 92−94 °C. HRMS (ESI-FT-ICR) m/z: [M + Na] Calcd for

C H NSNa 342.129, found 342.125. H NMR (MeOD, 500

MHz): δ 7.43 (dt, 6H, J = 8.6, 2.4 Hz), 7.31 (t, 6H, J = 7.6 Hz), 7.24

(

t, 3H, J = 7.3 Hz), 2.46 (t, 2H, J = 7.0 Hz), 2.37 (t, 2H, J = 7.3 Hz).

C{ H} NMR (CDCl , 150 MHz): δ 145.1, 129.8, 128.0, 126.8, 66.7,

1.2, 36.4.

Synthesis of 2-{[(4-Methoxyphenyl)(diphenyl)methyl]sulfanyl}-

ethan-1-amine 6a. Cysteamine hydrochloride (1.136 g 10 mmol)

was dissolved in 5 mL of DMF in an Erlenmeyer ﬂask equipped with a

magnetic stirrer, and to the ﬂask was added 3.08 g (10 mmol) of 4-

methoxytrityl hydrochloride. The mixture was stirred at room

temperature for 3 h. The resulting solution was concentrated under

the stream of nitrogen, then neutralized with a 1 M NaOH water

solution to a pH of 9 and evaporated under the stream of nitrogen

overnight. The resulting mixture was extracted four times with DCM

μL of 70% HCOOH/H

under the stream of nitrogen and lyophilized. Then, it was dissolved

in 0.1% HCOOH in MeCN/H O (1:1) and measured by FT-ICR-

O overnight. The ﬁltrate was evaporated

MS. After proving the completeness of the synthesis and determining

the substitution level, the whole peptidyl resin was subjected to Fmoc

deprotection, and side protecting groups were removed using 1 mL of

(

4 × 25 mL), then all fractions were collected, washed with water (2

a mixture of TFA/H O/TIS/EDT (94:2:2:2) for 2 h. Then, the

25 mL) and brine (2 × 25 mL), dried over MgSO , and evaporated

solution was ﬁltered out, the peptidyl resin was washed three times

with DCM and neutralized with 5% DIPEA/DMF (three washing

steps for 5 min each), and the standard drying procedure was applied.

Automated Microwave-Assisted on-Resin Formation of N-

2-[Thioethyl]glycine and the Further Peptide Precursor.

Partially Automated Microwave-Assisted Synthesis. TentaGel MB

NH2 resin with incorporated N-2-[(trt)thioethyl]glycine-AAM or N-

2-[(trt)thioethyl]glycine-βAβAM was prepared manually as men-

tioned above and placed in the microwave reactor of the Biotage

Initiator+ Alstra peptide synthesizer. For the ﬁrst coupling, 6−8 equiv

of Fmoc-Aa, DIC, and Oxyma Pure were used two times for 10 min in

on a rotary evaporator. A very dense and viscous yellow-orange oil

was obtained, and several solvents for crystallization were tested

(

ethyl acetate, acetone, CHCl , DCM, THF, and butanol with n-

hexane); however, no crystal form appeared. The ﬁnal mixture was

puriﬁed by column chromatography on silica using 5% MeOH/

CHCl ; yield: 1.48 g (4.24 mmol, 42.35%). TLC: 5% MeOH/CHCl ,

rf = 0.19. HRMS m/z: [M + Na] Calcd for C H NOSNa 372.139,

found 372.133. H NMR (MeOD, 500 MHz): δ 7.45−7.42 (m, 4H),

.35−7.32 (m, 2H), 7.31−7.28 (m, 4H), 7.24−7.21 (m, 2H), 6.87−

.84 (m, 2H), 3.79 (s, 3H), 2.47 (t, 2H, J = 7.2 Hz), 2.39 (t, 2H, J =

.5 Hz); ¹³C{ H} was reported by Riddoch.

75°. For the next couplings, the excess was reduced to 5 equiv and the

reaction time to 5 min at once. All reagents were used in a

concentration of 0.2 M in DMF. For Fmoc deprotection, 4.5 mL of

20% PIP/DMF was added two times for 3 and 10 min at rt with

oscillating mixing.

Manual On-Resin Formation of N-2-[Thioethyl]glycine by

the Incorporation of Protected Cysteamines. In a syringe

reactor was placed 200 mg of resin, which was swelled for 30 min at

room temperature in DMF. For 1, 2, and 3a, TentaGel HL NH resin

(

0.56 mmol/g) was used; for 3, TentaGel HL NH resin (0.26 mmol/

Fully Automated Microwave-Assisted Synthesis. For the fully

automated synthesis of peptide precursors with N-2-[(trt)thioethyl]-

glycine with a linker, several conditions for S-trityl-cysteamine

incorporation were studied and are presented in Table S1 . The

tested sequence was AAAAA-N-2-[(trt)thioethyl]glycine-AAM on a

TentaGel MB NH2 solid support, which was cleaved by the reaction

g) was used; and for 4 and 5, TentaGel S NH resin (0.23 mmol/g)

was used. Additionally, 4 was also synthesized on ChemMatrix resin

(

0.4−0.6 mmol/g). A linker containing either AAM (for 1, 3, and 3a)

or a βAβAM-sequence (for 2, 4, and 5) was synthesized according to

the standard Fmoc-SPPS strategy. For the coupling, 3 equiv of TBTU

or PyBOP, 6 equiv of DIPEA, and 3 equiv of the corresponding

Fmoc-protected amino acid in 1 mL of DMF were poured into the

syringe, and the mixture was placed for 20 min in the ultrasonic bath.

After ﬁltrating and washing the peptidyl resin ﬁve times with DMF,

with 20 μL of 3 M BrCN/DCM plus 200 μL of 70% HCOOH/H O

overnight. For comparison, conditions for the fully manual and

partially automated syntheses are also listed.

Fmoc Substitution Level Determination. For the determi-

nation of the ε value for the dibenzofulvene-piperidine adduct, 7.77

mg of Fmoc−Phe−OH (20,05 μmol) was dissolved in 1 mL of 20%

PIP/DMF, and the mixture was incubated for 20 min at room

temperature. Then, the volume was adjusted to 10 mL, the

absorbance spectrum was recorded after dilution, and the reference

maximum of absorption was chosen as 290 nm according to the

5% PIP/DMF was poured into the mixture for Fmoc deprotection,

and the resin was stirred in the same way for 5 min. After this time,

the reagent was ﬁltered out, and the peptidyl resin was washed seven

times with DMF. Next, bromo- or chloroacetic acid was added (5

equiv) with 5 equiv of DIC in 1 mL of DMF three times. The mixture

was stirred for 30 min on a rotary mixer, followed by ﬁltrations and

washing seven times with DMF. Then, 6 equiv of the protected

cysteamine was added in 1 mL of DMF, and the mixture was stirred

overnight at room temperature on a rotary mixer. Next, the peptidyl

resin was ﬁltered and washed ﬁve times with DMF. The reaction steps

were monitored with both Kaiser and chloranil tests. After synthesis,

the peptidyl resin was washed with DMF/DCM, DCM, DCM/THF,

recommendations of Bachem. For comparison, we also recorded the

UV−vis spectra of Fmoc−OSu and Fmoc−Ala−OH, which were

treated in the same way. A series of dilutions was prepared, and the

absorption was measured for the calibration curve. Pure 20% PIP/

DMF was used as a blank. The determined extinction coeﬃcient was

ε₂₉₀_n_m= 5597.1 l/mol·cm, and ε₃₀₁_n_m= 6789.5 l/mol·cm. Then, 5 mg

of the examined peptidyl resin was swelled directly in 20% piperidine/

DMF for 20 min. After ﬁltration, the resin was washed three times

with 1 mL of the same solution, and all rinses were collected together

and diluted to 10 mL. Then, the sample was diluted 10 more times

and measured in a quartz cuvette, with 20% PIP/DMF a blanking.

The determination of the substitution level was repeated at least two

times. Substitution level was calculated from the ε₂₉₀value and

dilution factor according to the equation:

THF, and THF/Et O and dried in a desiccator. For monitoring, 10

mg of N-2-[(trt)thioethyl]glycine-AAM-TentaGel was incubated in

0 μL of 3 M BrCN/DCM plus 200 μL of 70% HCOOH/H O

overnight. The ﬁltrate was evaporated under the stream of nitrogen,

dissolved in water with 10% MeCN, and directly measured by LC-

UV-MS. rt for N-2-[(trt)thioethyl]glycine-AA-HL: 11.8−12 min (1−

0% B/A in 15 min). HRMS (ESI-IT-TOF) m/z: [M + H] Calcd for

C H N O S 603.2635, found 603.2639. rt for [N-2-[thioethyl]-

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Note

0⁵E

TentaGel MB NH resin −0.23 mmol/g). To conﬁrm the designed

^SFmoc[mmol/g] = ¹

structures, only 3 mg of peptidyl resins CAKPGG-N-2-[thioethyl]-

glycine-linker-resin were treated with cyanogen bromide (20 μL of 3

^ε²90m_r_e_s_i_n

Peptide Cyclization Procedures. Procedure A. Through double

on-resin transthioesteriﬁcation (MESNa then MPAA) (Table 2): 200

M BrCN/DCM plus 200 μL of TFA/H O/TIS (95:2.5:2.5)) to

cleave the whole peptide construct from the solid support. In the

second experiment, 5 mg of CAKPGG-N-2-[thioethyl]glycine-linker-

resin was subjected to cyclization Procedure C with no further

puriﬁcation. Each product was then dissolved in the same volume of

water and analyzed by LC-MS using identical injection volume. The

results are presented in SI 4 .

Optimization of the Transthioesteriﬁcation Reaction Time. The

AAAAA-N-2-[thioethyl]glycine-AAM-TentaGel peptide precursor

was synthesized partially automated. After incubating the portions

Table 2. Procedures Tested for the Peptide One-Pot

Cyclization

procedure external thiol

buﬀer/pH

on-resin or in-solution

on-resin

(1) MESNa

2)MPAA

(1) citrate/3

(2) phosphate/7.4

(1) citrate/3

(

MESNa

on-resin

(10 mg) of the peptidyl resin in citrate buﬀer (pH 3, 300 μL) with

(

2) phosphate/7.4

MESNa (50 equiv, 8.5 mg) after diﬀerent periods in a 40 °C water

bath, the ﬁltrates were collected, desalted by omix tips, lyophilized,

dissolved in 0.5 mL of water, and measured by LC-UV-ESI-MS.

Similarly, the remained resins were dried and treated with cyanogen

bromide (20 μL of 3 M BrCN/DCM plus 200 μL of 70% HCOOH/

citrate/3

TEAB/7.0

on-resin

in-solution

MPAA

H O overnight). The ﬁltrates were evaporated, lyophilized, dissolved

mg of resin with an unprotected peptide precursor was swelled in 1

mL of citrate buﬀer (pH 3). Then, corresponding to the resin loading,

in 1 mL of water, and measured in the same way. The graph showing

the percentage of the ﬁnal product in time is shown in Figure S16. For

the transthioesteriﬁcation reaction, the ﬁnal products are the H-

0 equiv of MESNa was added in a volume of citrate buﬀer that gave

the ﬁnal peptide concentration of 3 mmol. The reaction mixture was

incubated in a water bath (40 °C) for 24 h and mixed manually from

time to time. Next, an additional solution of 50 equiv of MPAA with

AAAAA-MESNa thioester (LC-UV-IT-TO;, rt = 4.8−4.9 min (1−

0% B/A in 15 min), HRMS (IT-TOF) m/z [M + H] Calcd for

C H N O S 498.1687, found 498.1693) and the unreacted peptidyl

8 2

0 equiv TCEP in the same amount of phosphate buﬀer (pH 7.4) was

resin AAAAA-N-2-[thioethyl]glycine-AA-HL (LC-UV; rt = 5.9−6.1

min).

Synthesized Peptides. For each peptide, the most eﬃcient

synthetic procedure is described in the preparative scale with

puriﬁcation, and the compared procedures are described on an

analytical scale. Some co- and byproducts are also described.

Synthesis of Cyclo-CFGPKA 1. CFGPKA-N-2-[thioethyl]glycine-

poured into the mixture. Then, the pH of the ﬁnal solution was

adjusted with 8 M NaOH to the value of 7.4, and the reaction mixture

was incubated at 40 °C for the next 24 h. After this time, the syringe

content was ﬁltered out and desalted by SPE. The fractions containing

the desired product were evaporated under the stream of nitrogen and

lyophilized.

Procedure B. Through a single on-resin transthioesteriﬁcation with

MESNa: 200 mg of resin with an unprotected peptide precursor was

swelled in 1 mL of citrate buﬀer (pH 3). Then, corresponding to the

resin loading, 50 equiv of MESNa was added in a volume of citrate

buﬀer that gave the ﬁnal peptide concentration of 3 mmol. The

reaction mixture was incubated in a water bath (40 °C) for 24 h and

mixed manually from time to time. To increase the reaction yield, the

next 10 equiv of DTT in phosphate buﬀer was added to the mixture.

The pH was adjusted to 7.4 with 8 M NaOH, and the syringe was

placed into the water bath for the next 24 h in 40 °C. After this time,

the syringe content was ﬁltered out and desalted by SPE. The

fractions containing the desired product were evaporated under the

stream of nitrogen and lyophilized.

Procedure B1. Self-cyclization without any additional thiol: 100 mg

of resin with the unprotected peptide precursor was incubated in 10

mL of citrate buﬀer at pH 3 with 10 equiv of DTT in the water bath

for 24 h at 50 °C, and the mixture was mixed manually from time to

time. After this time, the syringe content was ﬁltered out and desalted

by SPE. The fractions containing the desired product were evaporated

under the stream of nitrogen and lyophilized.

Procedure C. Through a single on-resin transthioesteriﬁcation with

MPAA: 20 mg of resin with the unprotected peptide precursor was

placed in an Eppendorf tube and suspended in a 200 μL solution of

AAM-TentaGel HL NH (400 mg, ﬁnal loading of 0.16 mmol/g) was

subjected to cyclization procedure B. The crude product was puriﬁed

by HPLC, yielding 10.21 mg of the peptide (white powder, 17 μmol,

cyclization yield of 25.8%, ﬁnal yield of 15.9%). LC-UV-MS: rt = 6.6−

.9 min (5−60% B/A in 15 min). HRMS (ESI-IT-TOF) m/z: [M +

H] Calcd for C H N O S 604.2911, found 604.2914.

28 42

Synthesis of the 2-Mercaptoethanesulfonate Thioester of

CFGPKA 1a. CFGPKA-N-2-[thioethyl]glycine-AAM-TentaGel HL

NH (5 mg) was subjected to the ﬁrst step of cyclization procedure

B and thus was only incubated with 50 equiv of MESNa in citrate

buﬀer (pH 3) for 24 h at 40 °C and desalted. HPLC: rt = 23.5−24.0

min (gradient: 0−80% B/A in 40 min). HRMS (ESI-FT-ICR) m/z:

[

M + H] for C H N O S Calcd 746.2670, found 746.2323. One

9 3

aliquot was incubated in deionized water for several days and

measured by Thermo analytical chromatography, and the second one

was incubated with 10 equiv of DTT in phosphate buﬀer (pH 7.4) at

0° at room temperature, desalted, and lyophilized.

Synthesis of [Cys ]-axinellin A 2. CFPNPFT-N-2-[thioethyl]-

glycine-AAM-TentaGel HL NH2 (20 mg, ﬁnal loading of 0.18 mmol/

g) was subjected to cyclization procedure C. The crude product was

puriﬁed by HPLC, yielding 1.37 mg of the peptide (white powder, 1.6

μmol, cyclization yield of 55.0%, ﬁnal yield of 38.1%). LC-MS: rt =

.08 min. (5−60% B/A in 15 min). HRMS (ESI-qTOF) m/z: [M +

0 equiv of MPAA and 15 equiv of DTT in 0.5 mL of 1 M TEAB

H] Calcd for C H N O S 807.349, found 807.354.

buﬀer, with the ﬁnal pH adjusted to 7. The obtained mixture was

heated at 50 °C over 48 h. After that time, a few droplets of

concentrated triﬂuoroacetic acid were added to acidify the solution.

This in turn resulted in the precipitation of MPAA, which was then

extracted with diethyl ether (3 × 50 μL). To remove the volatile

buﬀer, the mixture was lyophilized.

Procedure D. Through transthioesteriﬁcation in solution: 2 mg of

the linear peptide precursor was placed in an Eppendorf tube. The

next stages were carried out analogously as those described for

procedure C.

39 51

Synthesis of [D-Thr ,Cys ]axinellin A 2a. The synthesis of the

peptide precursor was performed in a partially automated way. For the

D-Thr coupling, 6 equivs of Fmoc−D-Thr(OtBu)−OH, DIC, and

Oxyma Pure were used and coupled twice within 10 min at 75° C. For

cyclization, 90 mg of resin with a precursor was subjected to

cyclization procedure C and measured by LCMS-9060 without

further puriﬁcation: rt = 8.72 min. (5−60% B/A in 15 min). HRMS

(

ESI-qTOF) m/z: [M + H] Calcd for C H N O S 807.349, found

39 51 8 9

07.354.

Synthesis of Cyclo-CPKA 3. CPKA-N-2-[thioethyl]glycine-AAM-

TentaGel MB NH (140 mg, ﬁnal loading: 0.21 mmol/g) was placed

Synthesis of the Model Peptide Cyclo-CAKPGG Containing

Diﬀerent Linkers and Study on Their Impact on the Final

Cyclization. Four diﬀerent linkers were synthesized manually. The

synthesis was carried out only on an analytical scale (20 mg of

in syringe tubes in 10 mg portions and subjected to cyclization

procedure B. The modiﬁcation was applied in the following amount of

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Complete contact information is available at:

Note

phosphate buﬀer: 10 mL was added to each syringe. After cyclization,

all fractions were collected together, desalted, and lyophilized. To the

crude product was added 0.5 mg of TCEP, and the mixture was

incubated for 2 h at 50° C and puriﬁed by preparative HPLC

Mateusz Waliczek − Faculty of Chemistry, University of

Wroclaw, 50-383 Wroclaw, Poland

Anna Dziadecka − Faculty of Chemistry, University of

Wroclaw, 50-383 Wroclaw, Poland

(

Varian), yielding in 2.83 mg of the peptide (yellow oil, 7.1 μmol,

cyclization yield of 24.1%, ﬁnal yield of 22.0%). HPLC: rt = 2.8−3.2

min (1% in 8 min, 1−10% in 10 min, 10% in 10 min, 10−100% in 6

min B/A). HRMS (ESI-IT-TOF) m/z: [M + H]⁺Calcd for

C H N O S 400.2013, found 400.2016.

https://pubs.acs.org/10.1021/acs.joc.1c01045

Notes

Synthesis of Cyclo-CPKA 3 with No Thiol Addition. 100 mg of

CPKA-N-[2-thioethyl]glycine-AAM-TentaGel NH2 (ﬁnal loading of

.21 mmol/g) was placed in portions in a syringe tube and subjected

The authors declare no competing ﬁnancial interest.

to the cyclization Procedure B1. The modiﬁcation was applied in the

amount of phosphate buﬀer: 10 mL was added. After 24h incubation,

the ﬁltrate was desalted, lyophilized, and analyzed by LCMS 9030

ACKNOWLEDGMENTS

■

This work was supported by Grant UMO-2019/35/N/ST4/

0477 “Application of N-(2-thioethyl)glycine analogues in

(

ESI-qTOF).

Synthesis of Bicyclo-CPKACPKA 3a. CPKA-N-2-[thioethyl]-

orthogonal cleavage of peptides from the solid support” from

the National Science Centre, Poland. The authors are also

grateful to Andrzej Reszka (SHIM-POL “A.M. BORZYMOW-

SKI”, Poland) for providing the following: the Shimadzu

LCMS-IT-TOF, the LCMS-9030, and the automated micro-

wave peptide synthesizer Biotage Initiator+ Alstra. The authors

are grateful to Prof. Krzysztof Rolka, Head Department of

Molecular Biochemistry at the University of Gdansk (Poland),

for providing the reference sample of SFTI-1. The authors

would like to express their gratitude to Dr. Vincent Aucagne,

the coordinator of the team of molecular, structural and

chemical biology, and Dr. Aromal Asokan from CBM, CNRS-

Orléans (France) for the critical proofreading and suggestions

on the manuscript.

glycine-AAM-TentaGel MB NH2 (400 mg, ﬁnal loading of 0.07

mmol/g) was subjected to cyclization procedure A. The crude

product was puriﬁed by HPLC, yielding in 2.27 mg of a white powder

peptide (28 μmol, cyclization yield of 20.23%, ﬁnal yield of 6.16%).

LC-UV-MS: rt = 5.2−5.5 min (1% in 5 min, 5−10% in 10 min, 10%

in 5 min, 10−100% in 3 min B/A). HRMS (ESI-IT-TOF) m/z: Calcd

_M₊₂_H_]₂+ for C H N O S 399.1935, found 399.1934.

[

34 58 10 8 2

Synthesis of SFTI-1 4. CTKSIPPICFPDGR-N-2-[thioethyl]-

glycine-AAM-TentaGel MB NH2 (20 mg, ﬁnal loading of 0.16

mmol/g) was subjected to cyclization procedure C. The crude

product was puriﬁed by HPLC, yielding in 1.9 mg of a white

powdered peptide (1.2 μmol, cyclization yield of 40%, ﬁnal yield of

7.8%). LC-UV: rt = 8.05 min. (5−65% B/A). HRMS (ESI-qTOF)

m/z: [M + 2H] Calcd for C H N O S 757.3684, found

106 18 18 2

57.368.

Synthesis of SFTI-1 4 with Cyclization In-Solution. CTKS-

IPPICFPDGR-N-2-[thioethyl]glycine-AAM-ChemMatrix (20 mg)

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