A Strategy to Prepare Peptide Heterodimers in the Solid Phase with an Acid-Labile Linker

Article abstract of DOI:10.1021/acs.orglett.9b02638

The currently synthetic methods of peptide heterodimer involve tedious synthesis and purification steps. An acid-labile traceless linker was prepared, which is highly compatible with the Fmoc strategy and could be used to prepare peptide heterodimer on re

Full text of DOI:10.1021/acs.orglett.9b02638

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
A Strategy To Prepare Peptide Heterodimers in the Solid Phase with
an Acid-Labile Linker
Gang Wang,† Tingting Chen,† Tao Peng,† Shouguo Zhang,† Junyi Wang,§ Xiaoxue Wen,†
Xiaoming Yang,*,‡ and Lin Wang*,†
†Beijing institute of Radiation Medicine, Beijing 100850, P.R. China
‡Beijing institute of Lifeomics, Beijing 102206, P.R. China
§College of Engineering, Peking University, Beijing 100871, P.R. China
S
* Supporting Information
ABSTRACT: The currently synthetic methods of peptide heterodimer involve tedious synthesis and purification steps. An
acid-labile traceless linker was prepared, which is highly compatible with the Fmoc strategy and could be used to prepare
peptide heterodimer on resin. The linker could be cleaved concomitantly with peptide cleavage, and two model heterodimers
were synthesized. The proposed synthesis procedure is simple, straightforward, and provides great convenience for preparing
disulfide-linked peptide heterodimers.
result in low yields.10,11 Thus, while this method was very
arious peptides and small proteins, such as insulin and
Vrelaxin, exist as heterodimers, which are attractive targets effective, its complexity emphasized the need for an improved
synthetic route to obtain heterodimeric peptides through fewer
synthetic steps.
for the development of next-generation therapeutics owing to
their pleiotropic physiological properties.1,2 Research on the
structure−function relationship in heterodimeric peptides
plays an important role in developing new lead compounds,3,4
and finding effective and selective approaches to achieve
heterodisulfide formation between two peptide chains is an
important area of research.
Patil12 reported a method that imitated the insulin synthesis
procedure in vivo cellular, whereby both chains are sequentially
synthesized on the same solid support and separated by a
chemically cleavable bis-linker tether. In this method, the two
peptide chains were conveniently synthesized, the disulfide
bridge was formed in solution, and the bis-linker tether was
removed with hydrazine buffer. However, this strategy also
presented several obvious drawbacks. First, 4-
(hydroxymethyl)benzamide in the tether linker13 is base labile
and prone to nucleophilic attack. More importantly, in this
method, the disulfide bonds are formed in solution. To ensure
the intramolecular disulfide formation, oxidation was per-
formed in very dilute solution. These processes render the
workup tedious.
Several methods to prepare heterodisulfide peptide dimers
have been published. The initial method involved the separate
assembly of two S-reduced chains and subsequent oxidative
folding in solution. However, this method produced randomly
formed heterodimers, which means the target product was not
always obtained. Thus, this method was limited in scope.5,6
Another important strategy to prepare heterodisulfide
peptide dimers involved the directed, stepwise formation of
disulfide bridges. This method activated the thiol group of one
peptide and then added the second peptide in its free thiol
form.3,7,8 Here, the target asymmetric heterodimer was formed
via thiol exchange in acidic conditions without formation of
homodimer. However, the two peptides must be prepared and
purified before they can be combined, which involves tedious
steps.9 This method also required orthogonal S-protection and
the solid-phase synthesis of the two separate chains followed
by their release from the solid support. Multistep solution-
phase reactions can produce the correct disulfide pairing but
In the present work, we develop an analogous strategy that is
simple, compatible, and straightforward for overcoming the
limitations described above and develop a facile synthesis
procedure. Our proposed procedure involves an acid-labile
linker and allows intramolecular coupling on resin. Two key
structures are essential to link two peptide chains together: one
Received: July 26, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b02638
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to anchor the N-terminal and another to anchor the C-
terminal. As tert-butyloxycarbonyl (Boc) and p-alkoxybenzyl
benzyl (linker of Wang resin) are widely used in peptide
synthesis and labile to acidic conditions, we synthesized a
linker containing these molecules. Similar to the Boc and p-
alkoxybenzyl alcohol, the designed linker is compatible with
the Fmoc-tBu SPPS strategy and most disulfide bridge-
formation conditions and also easily removed during peptide
acidic cleavage (Scheme 1).
Scheme 3. General Synthesis Procedure of Acid-Labile
Linker
Scheme 1. Structure of Acid-Labile Linker
effect of the benzene ring, a suitable length is necessary; here,
we used ethyl 4-bromobutyrate to give compound 3. The ester
terminal was transferred to tertiary alcohol 6 by reaction with
excess Grignard reagent CH3MgBr. Before reacting, the OH
groups in 4 were preprotected with DHP (3,4-2H-dihydropyr-
an) to give compound 5. The tertiary alcohol in 6 was then
activated to react with the free α-amino groups. Compound 6
was first considered to transfer to the corresponding p-
nitrophenyl carbonate, but the transformation encounter failed.
Finally, we found that imidazole carbonate 7 prepared from
tertiary alcohol 6 and carbonyl diimidazole could be obtained
in excellent yield. Surprisingly, 7 is quite stable and easy to
store.
To obtain a heterodimer, peptide A was first assembled via
the standard Fmoc-SPPS strategy with an acid-labile resin,
such as Wang resin. The tether linker was then anchored to the
free α-NH2 group, and the free OH was exposed. The C-
terminal amino acid of peptide B was subsequently anchored
to the OH group. Standard SPPS amino acid coupling was
carried out to complete the assembly of peptide B. The
intramolecular disulfide bond was next formed on resin as
shown in Scheme 2.
The linker is easy to prepare in high yield (Scheme 3). To
construct the molecular bridge, 4-hydroxybenzaldehyde was
used as the initial material. 4-Hydroxybenzaldehyde was
reacted with a halogen aliphatic carboxyl ester that is used to
form the Boc-like structure in the next step. To decrease the
Using the proposed method, various heterodimers could be
easily prepared, regardless of the bridge types (i.e., disulfide or
lactam). In the present case, we only demonstrate how to
prepare a disulfide-bridge type peptide heterodimer. To
illustrate the overall method, a short insulin fragment was
synthesized as a model by using polystyrene-divinylbenzene
Wang resin (0.56 mmol/g) as the solid support (Scheme 4)
and Fmoc-Cys (Acm)-OH as the Cys residue. The first step
involves the sequential assembly of chain A via the standard
Fmoc-SPPS procedure to give peptide resin 9a. Linker 7 was
anchored to the free α-NH2 of 9a to give peptide resin 9b
(Scheme 5). To anchor linker 7 to the α-NH2 in 9a, we first
dissolved 7 and TEA in CH2Cl2 with DMAP as the catalyst
and then allowed the solution to react with peptide resin 9a for
24 h. The Kaiser test gave a positive result, which means 7
either reacts with amino groups with a slow rate or does not
react at all. Other methods, such as DBU14 catalysis, also
yielded a negative result. Hence, 7 was transferred to an onium
salt to enhance its activity. CH3I15 and TfOMe16,17 have been
reported to be suitable reagents for this purpose; here, we used
TfOMe as an additive to transfer 7 to onium salt 8, which
reacts completely and immediately. The high reactivity of 8
could acylate free amino groups in 9a smoothly to give the
peptide resin 9b (Scheme 5). Onium salt 8, however, is not
stable, and its slow and continuous decomposition in solution
could observed. Hence, the onium salt solution should be used
immediately after preparation.
Scheme 2. Strategy for Preparing Peptide Heterodimers in
the Solid Phase via an Acid-Labile Linker
Next, the THP group of 9b was removed, and elongation
was continued through the OH group of the linker. Since
TsOH has been used as a deprotecting reagent for THP during
solid-phase peptide synthesis,18 this reagent was selected to
B
DOI: 10.1021/acs.orglett.9b02638
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that the TsOH solution does not affect commonly used acid-
labile side chain-protecting groups, but Cys (Trt), Cys (Mmt),
and His (Trt) were found to deprotect slowly when treated
with TsOH, so these amino acid residues were recommended
for use in chain B.
Scheme 4. Preparation of Insulin Fragments Containing
One Intermolecular Disulfide Bridge
a
The next procedure involves an esterification of the free OH
in linker. Similar to the common loading situation, the DIC-
DMAP method is effective for most common amino acids, but
for the Cys and His situation, the MSNT/MeIm method was
chosen to decrease the epimerization.19 As a benefit of the the
high reactivity of the acylation, the esterification was complete
with 10 equiv of Fmoc amino acid within 8 h. The completion
of esterification and THP deprotection was confirmed by the
Fmoc quantification loading test.19 Chain B was sequentially
assembled to give 9d. A small amount of 9d was subjected to
Fmoc removal, cleaved, and then analyzed by RP-HPLC. Two
sharp peaks with good purity were observed in Figure 1a,
Figure 1. RP-HPLC chromatograms data: (a) mixture of chain A and
chain B; (b) crude peptide I. CH3CN−H2O (0.1% TFA) as the eluent
system (1 mL/min, 20%−50%, 30 min).
which indicates that the acid-labile linker was removed and
that the two peptide chains were synthesized successfully. The
disulfide bond was formed on resin by using I2 in DMF20 for
15 min × 2. Finally, the peptide was cleaved by TFA/TIS/
EDT/m-cresol/H2O (90:2.5:2.5:2.5:2.5, v/v) solution. After
concomitant cleavage of the molecular bridge, the peptide
heterodimer was obtained. Crude peptide I was analyzed by
RP-HPLC, and a sharp peak was observed (11.6 min in Figure
1b). Two byproducts (8.5 and 13.5 min in Figure 1b) were
identified as two homodimers. This result was confirmed by
MS and reveals that the peptide heterodimer was successfully
obtained. The results also show that intermolecular coupling is
inevitable in the preparation of the intramolecular disulfide
bond by oxidation.
a
Reagents and conditions: (a) 10 equiv of Fmoc-AA-OH, 5 equiv of
DIC, 0.2 equiv of DMAP in CH2Cl2-DMF (9:1, v/v), 8 h; (b) 20%
piperidine−DMF, 15 min; (c) 4 equiv of Fmoc-AA-OH, 3.8 equiv of
HATU, 8 equiv of DIPEA in DMF, 2 h; (d) 8 equiv of 8, 10 equiv of
Et3N in anhydrous CH2Cl2, 12 h × 2; (e) 5 mg/mL of TsOH in
CH2Cl2−MeOH (9:1, v/v), 60 min × 2; (f) 5 equiv of I2 in DMF, 15
min × 2; (g) TFA/TIS/EDT/m-cresol/H2O (90:2.5:2.5:2.5:2.5, v/
v), 1 h.
Scheme 5. Loading of Linker 7 to the Peptide Resin 9a
The proposed strategy may also be employed to develop
more complicated heterodimers, which contains two disulfide
bonds. TD1 (ACSSSPSKHCG) is a transdermal peptide that
has been shown to facilitate efficient transdermal protein drug
delivery through intact skin.21 Oxytocin (OXT, CY-
IQNCPLG) is a peptide hormone and neuropeptide that is
known to affect a variety of social and nonsocial activities and
behaviors.22 Herein, we prepared a TD1-OXT (OXT as an
analogue with carboxyl acid in C-terminal) heterodimer by
using two intermolecular disulfide bridges via our method to
illusion the two-disulfide situation chemically (Scheme 6).
In order to show the compatibility of the traceless linker and
avoid the formation of homodimers, we considered the
nonoxidative solid-phase method to prepare the heterodimer
TD1-OXT, which was mediated by 2,2′-dithiobis(5-nitro-
pyridine) (DTNP).11,23 The OXT analogue was first
remove OH groups in this work. A mixture of p-TsOH (5 mg/
mL) in CH2Cl2−MeOH (9:1, v/v) was found to complete the
deprotection within 60 min × 2, giving peptide resin 9c. Note
C
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DMF for 10 h. The resulting resin 11b was reacted with 10
equiv of DTNP for 1 h, and the free thiol group was
reprotected with 5-nitropyridinsufenyl (5-Npys) to give resin
11c. Resin 11c was then treated with dilute TFA solution
(TFA/TIS/CH2Cl2 = 1:5:94, v/v) for 6 × 3 min to remove the
Mmt group in chain B. Thereafter, the 5-Npys group was
protonated and activated to attack the free thiol group. The
first disulfide bond was completed after further agitation in
DMF for 1 h. The peptide was then cleaved by TFA solution.
The resulting crude peptide was subsequently purified and
analyzed by RP-HPLC (Figure 2). The purified 11e was then
diluted with acetic acid prior to treatment with excess iodine
(I2) to form the second disulfide (Figure 3).
Scheme 6. Preparation of TD1-OXT Peptide Containing
Two Intermolecular Disulfide Bridges
a
Figure 2. RP-HPLC chromatograms data: (a) microcleavage of
peptide−resin 11a and analysis; (b) microcleavage of peptide−resin
11b and analysis; (c) crude 11e. CH3CN−H2O (0.1% TFA) as the
eluent system (1 mL/min, 10%−40%, 30 min).
a
Reagents and conditions: (a) 10 equiv of Fmoc-AA-OH, 5 equiv of
DIC, 0.2 equiv of DMAP in CH2Cl2−DMF (9:1, v/v), 8 h; (b) 20%
piperidine−DMF, 15 min; (c) 4 equiv of Fmoc-AA-OH, 3.8 equiv of
HATU, 8 equiv of DIPEA in DMF, 2 h; (d) 8 equiv of 8, 10 equiv
Et3N in anhydrous CH2Cl2, 12 h × 2; (e) 5 mg/mL of TsOH in
CH2Cl2−MeOH (9:1, v/v), 60 min ×2; (f) 20% BME in DMF, 0.1 M
DIPEA, 10 h; (g) 10 equiv of DTNP in CH2Cl2, 1 h; (h) TFA/TIS/
CH2Cl2 (1:5:94, v/v), 6 × 3 min, then in DMF for 1 h; (i) TFA/TIS/
EDT/m-cresol/H2O (90:2.5:2.5:2.5:2.5, v/v), 1 h; j) I2 in HOAc−
H2O (4:1, v/v).
Figure 3. RP-HPLC chromatograms data during I2 oxidation: (a)
purified 11e (dissolved in HOAc−H2O = 4:1, v/v); (b) peptide II.
CH3CN−H2O (0.1% TFA) as the eluent system (1 mL/min, 10%−
40%, 30 min).
In conclusion, we offer a new approach to prepare peptide
heterodimers containing intermolecular disulfide bridges with
the assistance of an acid-labile linker. Both oxidation and
nonoxidation methods can match the strategy effectively. The
proposed method is highly compatible with the Fmoc strategy
and, therefore, could be used in many applications. More
importantly, the linker is easily prepared and extremely stable.
Using our method, two heterodimers were synthesized, and
intermolecular disulfide bonds between the two peptide chains
were formed on resin in one pot, thereby minimizing the need
for tedious synthesis and purification steps.
assembled as chain A and TD1 as chain B. To prepare the
heterodimer peptide, Cys should be orthogonally protected
and the disulfide bonds precisely matched. StBu-Acm was used
to protect chain A, while Mmt-Acm was used to protect chain
B. The disulfide bond close to the N-terminals of A and B was
considered to form first to avoid the steric hindrance of the
solid phase. The StBu group in chain A was removed by
treating the peptide-resin with 0.1 M DIPEA in 20% BME/
D
DOI: 10.1021/acs.orglett.9b02638
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(17) Roychoudhury, R.; Pohl, N. L. B. Org. Lett. 2014, 16 (4),
1156−1159.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b02638.
■
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Detailed experimental procedures, characterizations, and
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AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: wanglin@ bmi.ac.cn.
*E-mail: xiaomingyang@sina.com.
ORCID
Lin Wang: 0000-0001-8721-2038
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the China Postdoctoral Science
Foundation General Grant (2016M592978), Special Grant
(2017T100816), and National Science Foundation of China
(Grant No. 81273431).
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