Use of solid-supported 4-fluorophenyl 3-nitro-2-pyridinesulfenate in the construction of disulfide-linked hybrid molecules

Article abstract of DOI:10.1039/d0ob01370f

To construct disulfide-linked hybrid molecules systematically and efficiently, we established a more practical solid-phase disulfide ligation (SPDSL) system with enhanced utility. The group Npys-OPh(pF) shows reactivity similar to that of Npys-Cl, but it is more stable. An efficient synthesis of the cyclic peptide oxytocin and a peptide-sugar conjugate was accomplished as models. These results indicate that the Npys-OPh(pF) resin functions as a common synthetic platform in SPDSL.

Full text of DOI:10.1039/d0ob01370f

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Use of solid-supported 4-fluorophenyl 3-nitro-2-
pyridinesulfenate in the construction of disulfide-
linked hybrid molecules†
Cite this: DOI: 10.1039/d0ob01370f
Received 4th July 2020,
Accepted 7th September 2020
Yan Cui, Akihiro Taguchi, Kiyotaka Kobayashi, Hayate Shida, Kentaro Takayama,
DOI: 10.1039/d0ob01370f
Atsuhiko Taniguchi and Yoshio Hayashi
*
rsc.li/obc
To construct disulfide-linked hybrid molecules systematically and the two-step reactions shown in Fig. 1B. The two steps include
efficiently, we established a more practical solid-phase disulfide the following: (1) the t-Bu protected Cys-containing derivative
ligation (SPDSL) system with enhanced utility. The group Npys- A is loaded onto the Npys-Cl resin (1) via an active disulfide
OPh(pF) shows reactivity similar to that of Npys-Cl, but it is more bond, and (2) component A on the resin is readily transferred
stable. An efficient synthesis of the cyclic peptide oxytocin and a to another unprotected SH-containing component B by a rapid
peptide–sugar conjugate was accomplished as models. These and selective disulfide exchange reaction, resulting in the for-
results indicate that the Npys-OPh(pF) resin functions as
a
mation of a disulfide conjugate (A-S-S-B). This strategy over-
comes drawbacks in the usual solution strategy such as
tedious purifications. This SPDSL strategy has been success-
fully applied to disulfide-driven cyclic peptide synthesis
(DdCPS) as a means of synthesizing cyclic disulfide peptides.5
In this DdCPS, after the preparation of the disulfide peptide,
the entropically advantageous intramolecular amide bond for-
mation can be achieved with the proximity effect of each reac-
tion point, and affords a promising method for the synthesis
of cyclic multi-disulfide peptides.5b This SPDSL strategy has
also achieved an efficient synthesis of peptide–drug conju-
gates.6 However, a major drawback of the system is that the
Npys-Cl resin (1) is unstable in the presence of light or moist-
ure and tends to dimerize even at low temperatures.7
Consequently, it must be prepared from its Bn form immedi-
ately prior to use.
common synthetic platform in SPDSL.
The disulfide bond is an important bond which stabilizes
three-dimensional structures and thus maintains the biologi-
cal functions of many peptides and proteins. It is also impor-
tant in the construction of valuable hybrid molecules.1 Two
thiol-containing units in bioactive molecules such as peptides,
proteins, sugars, nucleic acids or drugs can be selectively con-
nected by a disulfide link, and since the resultant hybrid mole-
cules express bifunctional actions, they can highly regulate the
biological systems in a coordinated manner. Hybrid molecules
have been used extensively in recent life science research pro-
grams, including drug discovery.2
There is however no integrated system built on the basis of
a common synthetic platform for the efficient construction of
disulfide-linked hybrid molecules. Thus, we focused on some
properties of the thiol protection afforded by the 3-nitro-2-pyri-
dinesulfenyl (Npys) group. This protective group was originally
developed by Matsueda and Aiba in 1978 and their first
reagent for protection was Npys chloride (Npys-Cl).3 Npys-pro-
tected cysteine (Cys(Npys)) is prepared with Npys-Cl and it is
highly reactive to unprotected thiol groups, with which it
forms the corresponding disulfide product.4 Focusing on the
chemoselectivity of the Npys group, we developed a one-pot
solid-phase disulfide ligation (SPDSL) system. This system
uses the Npys-Cl resin as a key chemical (Fig. 1)5 and involves
Department of Medicinal Chemistry School of Pharmacy, Tokyo University of
Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
E-mail: yhayashi@toyaku.ac.jp
Fig. 1 Development of the novel Npys-OPh(pF) resin (2) to replace the
unstable Npys-Cl resin (1) (A) and a schematic illustration of the Npys-
mediated solid-phase disulfide ligation (SPDSL) strategy (B).
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0ob01370f
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Since we had recently synthesized various alkyl and aryl tron-donating and electron-withdrawing groups at the p-posi-
3-nitro-2-pyridinesulfenates (Npys-OR) in order to develop a tion of the phenyl ring were examined. Introduction of elec-
new oxidative agent for the intramolecular disulfide for- tron-donating methyl or methoxy groups (entries 4 and 5) at
mation,8 we examined Npys-OR, which can be adapted to the this position led to slightly decreased yields (86% and 82%,
SPDSL strategy in place of Npys-Cl. It was found that the respectively). This is probably due to the slight increase of the
4-fluoro-phenoxide of Npys, i.e., Npys-OPh(pF), can serve as a pKa values of both leaving groups (pKa: 10.1 and 10.2,10
potential surrogate in the solution reaction. The corres- respectively) and the reduction of the electrophilic nature of
ponding Npys-OPh(pF) resin (2) was synthesized and its ability the sulfenate sulfur atom by increased electron donation from
as a surrogate was evaluated by its use in the synthesis of the the substituted phenyl ring. Conversely, when an electron-
mono-cyclic disulfide peptide and a peptide–monosaccharide withdrawing chlorine or fluorine atom was introduced at the
conjugate with a disulfide linkage. Its physicochemical stabi- p-position (entries 6 and 7; pKa of p-chlorophenol and p-fluoro-
lity was compared to that of the conventional Npys-Cl resin (1). phenol = 9.41 and 9.89 (ref. 10) respectively), the yields
In the development of a new solid-supported Npys agent improved, attaining values similar to those with Npys-Cl (98
applicable to the SPDSL strategy, the interaction of Npys and 96%, respectively). Based on these results, we selected
derivatives and S-protected cysteine derivatives is critical. p-fluorophenyl 3-nitro-2-pyridinesulfenate (Npys-OPh(pF)) as a
Accordingly, we evaluated the behavior of several newly devel- replacement for Npys-Cl.
oped Npys-OR compounds9 in the conversion reaction in a
In an effort to understand the effect of the solvent on the
solution of Fmoc-Cys(t-Bu)-OH to an active disulfide-contain- yield of the active disulfide, we examined the reaction in other
ing Fmoc-Cys(Npys)-OH. When Npys-Cl (1.2 eq.) was used, the solvent systems. In glacial acetic acid under anhydrous con-
desired compound was obtained in 97% yield under the afore- ditions, the reaction proceeded at rt, giving the product with a
mentioned reaction conditions with a 90% aqueous HCOOH good yield (97%, entry 8). However, in dichloromethane
solution as the solvent. When Npys-OMe (1.2 eq., entry 1), a (DCM), the reaction proceeded relatively slowly, giving a 66%
mild disulfide bond forming agent for unprotected Cys-con- yield (entry 9) and none of the desired product was obtained
taining peptides,8 was used, a moderate yield (57%) was under basic conditions with N,N-diisopropylethylamine
obtained at room temperature (rt) as can be seen in Table 1. (DIPEA) in DCM (entry 10). These results suggest that the reac-
Npys-OBn (1.2 eq., entry 2) gave a similar yield (59%) at 0 °C. tion proceeds under neutral or acidic conditions and is pro-
These results indicate that alkoxides with a pKa value of ∼15 moted in the presence of an acid. To further understand the
(ref. 10) are less reactive than Npys-Cl. With the use of a better reactivity of Npys-OPh(pF) with thiols and other thiol-protect-
leaving group, e.g. phenoxide (entry 3, Npys-OPh, pKa = 10 (ref. ing groups, several Fmoc-Cys(R)-OH derivatives were reacted
10)), the yield was improved to 88%. Accordingly, to adjust the with Npys-OPh(pF) under the formic acidic conditions defined
reactivity of the phenoxide further, compounds with both elec- above (Table S1, ESI†). As a result, the thiols protected by t-Bu,
Acm or 4-MeOBn are suitable for the formation of an active di-
sulfide. Finally, we chose the combination of Npys-OPh(pF)
and Cys(t-Bu) to realize SPDSL in the next experiment, which
Table 1 Synthesis of Fmoc-Cys(Npys)-OH from Fmoc-Cys(t-Bu)-OH
was performed on a solid-phase system.
and Npys-OR
To apply the chemistry of Npys-OPh(pF) to the SPDSL strat-
egy, we synthesized a solid-supported form of the compound.
As a solid support, a polyethylene glycol-based ChemMatrix®
resin,11 which swells in both organic and aqueous solutions,
was chosen. As shown in Scheme 1, the ChemMatrix® resin
with 3-nitro-2-benzylthiopyridine-5-carboamine (3) was con-
Conditions
Solvent
verted to the corresponding Npys-Cl resin by treatment with
2% (v/v) SO2Cl2/1,2-DCE in the presence of pyridine.5 The sub-
sequent reaction with 4-fluorophenol, producing the Npys-OPh
(pF) resin, was examined. The conversion efficiency was deter-
mined from the level of fluorine contained in the resin as
determined by elemental analysis. As shown in Table S2 (in
Temp.
(°C)
Time Isolated
Entry R1 a,b
(h)
yield (%)
1
2
3
4
5
6
7
8
9
10
Me
Bn
Ph
4-Me-Ph
90% HCOOH aq.
90% HCOOH aq.
90% HCOOH aq.
90% HCOOH aq.
rt
0
0
0
0
0
0
rt
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
57
59
88
86
82
98
96
97
4-MeO-Ph 90% HCOOH aq.
4-Cl-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
90% HCOOH aq.
90% HCOOH aq.
CH3COOH
DCM
0 to rtc 12
66
DCM, DIPEA (2 eq.) 0 to rtc 12
N.O.d
a The amount of Npys-OR is 1.2 equivalents. b Fmoc-Cys(Npys)-OH was
also synthesized in a yield of 97% with Npys-Cl in 90% HCOOH aq. at
0 °C for 30 min. c Reaction conditions: at 0 °C for 1 h and then at rt for
12 h. d N.O.: not obtained.
Scheme 1 Synthesis of the Npys-OPh(pF) resin (2).
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Table 2 Loading yields of peptide 4 onto the Npys-OPh(pF) resin
the ESI†), the best conversion efficiency of 75% was obtained
by a 10 min treatment with 4-fluorophenol (10 eq.) at rt in the
presence of DIPEA (8 eq.) as a base in 1,2-dichloroethane (1,2-
DCE), reaction conditions similar to those used in the solu-
tion-phase synthesis.9 In this way, the Npys-OPh(pF) resin is
easily prepared from available solid supports.
To confirm that the Npys-OPh(pF) resin is compatible with
the SPDSL strategy, a model study was carried out in the syn-
thesis of oxytocin. The resin was used in the synthesis of a di-
sulfide peptide consisting of two oxytocin-derived fragments,
the C-terminal Cys-protected 5-mer, H-Asn-Cys(t-Bu)-Pro-Leu-
Gly-NH2 (4), and the N-terminal Cys-free 4-mer, Fmoc-Cys-Tyr-
Ile-Gln-OH (5), which are intermediates in the synthesis of oxy-
tocin.5a As shown in Scheme 2, the resin was mixed with the
Cys(t-Bu) peptide (4), vortexed for 1–24 h and then washed
with CH3CN and H2O. The completion of the loading was indi-
cated by the disappearance of the peptide (4) from the solu-
tion, as monitored by HPLC analysis. As shown in Table 2,
Loading yield of
4a (%)
Resin 2
Entry Solvent
(eq.)
1 h 3 h
16
12 33
17 41
24 43
24 h
1
2
3
4
5
6
7
90% formic acid aq.
2.2
3.7
7.5
3.7
3.7
3.7
3.7
0
N.T.
N.T.
93
91
0
33
74
90% formic acid aq.
90% formic acid aq.
Acetic acid
CH3CN : H2O (1 : 1)
DMF
0.4 M LiCl/90% formic acid
aq.
0
0
0
0
55 60
8
0.4 M LiCl/acetic acid
3.7
99 N.
T.
N.T.
a Loading yield (%) = [1 − (HPLC peak area of residual peptide 4/HPLC
peak area of original peptide 4)] × 100. N.T.: not tested.
90% aqueous formic acid (entries 1–3) was first used, based on ethylene glycol-based Npys-ChemMatrix resin. Thus, an Npys-
the result from model study performed in solution OPh(pF) moiety can react efficiently with S-protected Cys-con-
a
(Table 1). With 2.2 eq. of resin, a low loading yield (16%) was taining peptides under acidic conditions on the resin, result-
observed in a 3 h reaction (entry 1), indicating that the Npys- ing in the formation of a solid-supported Npys-based active
OPh(pF) resin can load the peptide via an active disulfide. To disulfide.
optimize the reaction conditions, the amount of resin used
To construct a disulfide peptide, the resulting resin-bound
was studied (entries 2 and 3) and a high peptide loading yield C-terminal peptide fragment was transferred to another Cys-
(93%) was achieved with 7.5 eq. of resin and a vortex time of containing N-terminal fragment (5), whose N-terminal
24 h (entry 3). The reaction was further improved by the use of α-amino group was attached to an Fmoc-protecting group, by
acetic acid as a solvent which gave a similar peptide-loading vortexing the peptide-resin (6) with peptide 5 in DMF : H2O
yield (91%) in spite of the decreased quantity (3.7 eq.) of the (2 : 1) for 30 min at rt. The result was complete consumption
resin. Under non-acidic conditions, e.g. CH3CN : H2O (1 : 1) of peptide 5 and appearance of a single new peak, the desired
and DMF (entries 5 and 6), the loading was unsatisfactory with disulfide peptide (7), as observed in the HPLC analysis of the
yields of 0% and 33%, respectively, but upon the addition of reaction solution (for the HPLC chart, see Fig. S2, ESI†). After
LiCl (entries 7 and 8), the compounds were completely loaded removal of the resin by filtration, and with no further purifi-
onto 3.7 eq. of resin in 1 h (HPLC chart, see Fig. S1, ESI†).
cation steps, the disulfide peptide with 94% purity was
Since it has been reported that Li salts in an organic obtained from the filtrate with an isolated yield of 73%. This
solvent can not only change the conformation of a peptide by result is a formal total synthesis of oxytocin.5a As previously
complexation with an oxygen atom of each amide bond but reported,5a this disulfide peptide can be converted to oxytocin
also can swell the polystyrene resin,12 these effects might be through intramolecular amide formation and subsequent
also involved in the reaction between the peptide and poly- Fmoc deprotection.
To demonstrate the broad versatility of the reaction, the
synthesis of an Npys-OPh(pF) resin sugar hybrid molecule was
conducted. A reported disulfide linked glycopeptide, in which
a
pentapeptide of a human IgG2 sequence and an
N-acetylglucosamine derivative are linked by a disulfide bond,
was chosen. This compound was developed as a structural
mimetic of the natural asparagine glycosylate.13 A cumber-
some solution-phase synthesis of this compound was pre-
viously accomplished and included the preparation of acti-
vated thioglycoside, and the formation of a disulfide bond
between Cys-containing pentapeptide and thioglycoside. This
synthetic method of a glycoconjugate required three purifi-
cation steps. However, as shown in Scheme 3, our Npys-OPh
(pF) resin-mediated SPDSL of the pentapeptide (Ac-Phe-Cys(t-
Bu)-Ser-Thr-Phe-NH2) (8) formed a human IgG2 sequence with
Cys in place of Asn-297. A subsequent reaction with 2-acet-
Scheme 2 Synthesis of a disulfide bond in oxytocin via SPDSL with the
Npys-OPh(pF) resin (2). a With the final intramolecular amide formation
and Fmoc deprotection, oxytocin was successfully obtained.5a
amido-2-deoxy-1-thio-β-D-glucose (9) afforded
a disulfide-
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Acknowledgements
The authors acknowledge Mr H. Fukaya of Tokyo University of
Pharmacy and Life Sciences for mass spectral analysis. This
work was supported by the Japan Society for the Promotion of
Science (JSPS), KAKENHI, a Grant-in-Aid for Young Scientists
(B) 16K18914, Early-Career Scientists 19K16324 and Scientific
Research (B) 19H03356, Basic Science and Platform
Technology Program for Innovative Biological Medicine
(AMED, JP18am0301006) and MEXT-supported program for
the Private University Research Branding Project.
Scheme 3 Synthesis of disulfide-linked glycoconjugate 10 via SPDSL
with the Npys-OPh(pF) resin (2).
linked glycoconjugate (10) with 96% purity and an isolated
yield of 47%. In this case, only one final HPLC purification Notes and references
was necessary (see the ESI and Fig. S3 and S4† for details).
1 H. Choi, M. T. Jeena, L. Palanikumar, Y. Jeong, S. Park,
This result indicates that our SPDSL strategy using the Npys-
OPh(pF) resin can simplify the procedure, creating a disulfide-
linked conjugate not only between peptides but also between
sugars, and presumably, drugs, polynucleotides and proteins.
Finally, to demonstrate the usefulness of the Npys-OPh(pF)
resin, we investigated its long-term stability. As shown in
Table S3,† using the Npys-Cl resin after storage for 1 day at rt,
the loading of the peptide fragment failed. By contrast, the
Npys-OPh(pF) resin was largely unchanged after storage for
one day at rt, and gradually decomposed in one week
(Table S3, Fig. S5 and S6†). However, the Npys-OPh(pF) resin
was stable at −20 °C for more than 3 months (Table S3†).
These results indicate that the stored Npys-OPh(pF) resin is
more stable than the conventional Npys-Cl resin and the
preparation of the Npys-OPh(pF) resin immediately prior to its
use is unnecessary. This stands in contrast to the behavior of
the Npys-Cl resin.
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(2), a new Npys-mediated solid-support agent. This new agent
(2) can be prepared from the ChemMatrix® resin and 3-nitro-
2-benzylthio-pyridine-5-carboamine (3) with a straightforward
method in a short time. In the SPDSL strategy, the Npys-OPh
(pF) resin is applicable to disulfide bond formation in, for
example, the synthesis of oxytocin and disulfide-linked glyco-
conjugates. Moreover, the agent is more stable upon storage
than the conventional Npys-Cl resin (1) and as a result, the
Npys-OPh(pF) resin obviates a laborious activation step necess-
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SPDSL agent, the Npys-OPh(pF) resin can contribute to the
preparation of a variety of disulfide-linked conjugations useful
in peptide science, chemical biology and drug development.
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Conflicts of interest
There are no conflicts to declare.
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