Pyroacm Resin: An Acetamidomethyl Derived Resin for Solid Phase Synthesis of Peptides through Side Chain Anchoring of C-Terminal Cysteine Residues

Article abstract of DOI:10.1021/acs.orglett.6b00115

The design, synthesis and utilization of an efficient acetamidomethyl derived resin for the peptide synthesis is presented using established Fmoc and Boc protocols via side chain anchoring. Cleavage of the target peptide from the resin is performed using carboxymethylsulfenyl chloride under mild conditions which gave in situ thiol-sulfenyl protection of the cysteine residues. The utility of the resin is successfully demonstrated through applications to the syntheses of model peptides and natural products Riparin 1.1 and Riparin 1.2. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.orglett.6b00115

Letter
pubs.acs.org/OrgLett
Pyroacm Resin: An Acetamidomethyl Derived Resin for Solid Phase
Synthesis of Peptides through Side Chain Anchoring of C‑Terminal
Cysteine Residues
Vinayak Juvekar and Young Dae Gong*
*Innovative Drug Library Research Center, Dongguk University, Seoul 100-715, South Korea
S
* Supporting Information
ABSTRACT: The design, synthesis and utilization of an
efficient acetamidomethyl derived resin for the peptide synthesis
is presented using established Fmoc and Boc protocols via side
chain anchoring. Cleavage of the target peptide from the resin is
performed using carboxymethylsulfenyl chloride under mild
conditions which gave in situ thiol-sulfenyl protection of the
cysteine residues. The utility of the resin is successfully
demonstrated through applications to the syntheses of model
peptides and natural products Riparin 1.1 and Riparin 1.2.
eptides containing a C-terminal cysteine and cyclic
disulfide are highly abundant in nature and are employed
(1c),5b Hgm (1d2),5c Hqm (1d3),5c phthalimidomethyl
(1d1),5d and trifluoroactamidomethyl (Figure 1, 1e).5e
P
as valuable biological probes.1 Following its development by
Merrifield, solid phase peptide synthesis (SPPS) has been
employed for industrial and laboratory scale syntheses of
numerous synthetic peptides and proteins.2 Unlike almost all of
the other side chain functionality found in the 20 naturally
occurring amino acids utilized in SPPS, the free thiol in cysteine
is highly reactive and typically requires a trityl (Trt) or
acetamidomethyl (Acm, Figure 1, 1a) protection during
Despite these advances, the main challenge associated with
protection of C-terminal cysteine residues results from their
ready β-elimination and racemization reactions, which result in
formation of byproducts during each Fmoc SPPS coupling
cycle.6 To prevent β-elimination, side chain anchoring
techniques have been devised. One involves linking a terminal
cysteine to a trityl,7a,d 2-chlorotrityl,7b or Xal7c resin through a
thioether bond and the other to a masked carboxylic group at
the C-terminal.7e Unfortunately, the linkers and resins currently
employed for side chain immobilization are acid labile resulting
in partial cleavage under conditions that induce deprotection of
other protected amino acid residues.
To facilitate pre- or postcleavage, side chain modification of
other amino acid residues such as those required for installation
of a fluorophore or an oligo-saccharide, an analogue of the Acm
linker for cysteine thiol protection, is needed, and the resulting
process needs to be compatible with both the Fmoc and Boc
protocols. Unfortunately, the Acm group and most of its
relatives require Hg2+, Ag+, or Tl3+ salts for deprotection.3b,4,8
These toxic heavy metals are hazardous to the environment.
Moreover, it is difficult to remove thiol-metal complexes from
product mixtures. Carboxymethylsulfenyl chloride (ScmCl) is a
reagent that serves as an inexpensive and viable alternative for
Acm group removal.9a Moreover, the cleavage process leads to
in situ formation of a sulfenyl group protection at cysteine thiol.
Despite its potential advantages, this process has only been
explored using fully protected peptides in both solid and
solution phase synthesis.9 As a part of our high-throughput
solid-phase synthesis program,10 we required the availability of
Figure 1. Structures of acetamidomethyl 1a and its modified
protective groups.
peptide chain elongation.3 Whereas the Trt protective group
is acid labile, the Acm group can tolerate both acidic and basic
conditions ranging from hydrofluoric acid to hydrazine.
Moreover, it is stable under chemical ligation, desulfurization
and various reduction conditions including Zn in AcOH.3b,4
The stability of the Acm group makes it compatible with
manipulations used in tert-butyloxycarbonyl (Boc)/benzyl (Bn)
and fluorenylmethyloxycarbonyl (Fmoc)/t-Butyl (tBu) guided
peptide assembly and cleavage. Furthermore, the Acm group
has been modified to produce even more stable or orthogonal
thiol protecting groups. The modifications include trimethyla-
cetamidomethyl group (1b),5a S-phenylacetamidomethyl
Received: January 13, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b00115
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Letter
a facile and rapid procedure for preparation of cysteine
containing peptide and peptidometic molecules.
protected cysteine residue on resin 3a was explored using
Fmoc-L-Cys(Trt)-OAllyl (4a1), Fmoc-D-Cys(Trt)-OAllyl (4a2),
and Boc-L-Cys(Trt)-OAllyl (4b), prepared by allylation of
commercially available substrates (see Supporting Information
(SI)). Pyroacm resin 3a was subjected to trans-thioetherifica-
tion by using 3.0 equiv of 4a1 under acidic conditions. Optimal
conditions for this process (Table S2), which yields thioether
5a1, were found to be 15 vol % of TFA in CH2Cl2 for 2 h
(Scheme 2). UV spectroscopic analysis of the fluorenylmethyl
derivative of 5a1, generated by Fmoc cleavage (20% piperidine
in DMF), indicates that the resin loading is approximate 0.85
mmol/g. The optimal conditions were used to link 4a2 and 4b
to 3a, processes which lead to formation of 5a2 and 5b,
respectively. In the case of 5b, the extent of loading was
determined using quantitative Ninhydrin analysis.
Herein, we designed and synthesized a mimic of the Acm
linker, termed Pyroacm, which can be employed to anchor
terminal cysteines via the side chain thiol moiety. In addition,
we developed cleavage strategies for pre- and postmodifications
of peptides.
We hypothesized that it should be possible to couple 1-
(methoxymethyl)pyrrolidin-2-one (2a) or (2-oxopyrrolidin-1-
yl)methyl acetate (2b) to functionalized resins using C−C
bond forming enolate-alkylation reactions. In addition, we
believed that coupling of the cysteine residue to the Pyroacm
moiety on the resin would take place through thioether bond
formation under acidic conditions (Scheme 1).
Introduction of the cysteine residue on the Pyroacm resin
under acidic condition minimizes the risk of racemization at the
C-terminal residue. However, in the Fmoc protocol for SPPS,
repetitive treatment with a 20 vol % piperidine may induce
racemization of the cysteine residue. To examine the extent of
racemization occurring at the cysteine residue, Pyroacm resins
containing linked model tripeptide 7a1 and 7a2 were prepared
by reactions of 3a with the D and L isomers of Fmoc-Cys(Trt)-
OAllyl and Boc-L-Cys(Trt)-OAllyl, respectively, using Fmoc
and Boc protocols (Scheme 3). Sequential removal of the allyl
Scheme 1. Preparative Route for Implementation of
Pyroacm Based Strategy for Immobilization via a C-
Terminal Cysteine Residue
a
a
Reagent and conditions: (a) Installment of the Pyroacm linker on
standard Merrifield resin using nucleophilic displacement with enolate
of 2a or 2b. (b) Immobilization of the initial cysteine residue under
acidic conditions.
Scheme 3. Model Tripeptide Synthesis Utilizing the
Pyroacm Resin and Its Racemization Studies
To evaluate this proposal, we first prepared two Pyroacm
functionalized linkers 2a and 2b. The syntheses began with the
treatment of commercially available pyrrolidin-2-one with
formalin in the presence of KOH (Scheme 2). Subsequent
Scheme 2. Synthesis of the Pyroacm Resin and Trans-
Thioetherification of Cysteine Derivative onto Pyroacm
Resin
and N-protecting groups gave 7a1 and 7a2. Cleavage from the
resin to generate tripeptides 8a1 and 8a2 was performed by
treatment of 7a1 and 7a2 with 2 equiv of ScmCl in AcOH. The
diastereomers of 8a1 (Rt = 31.3 min) and 8a2 (Rt = 33.6 min)
can be readily separated using HPLC. Analysis of the
chromatograms revealed less than 2% epimerized products
8a1 and 8a2 are produced during Fmoc elongation protocol and
negligible amount of epimerized product 8a1, i.e., <0.4% during
Boc protocol (see SI).
methylation of the formed carbinolimide with MeI in THF
containing NaH gave the methyl ether derivative 2a (84%),
whereas acetylation of this intermediate produced the ester 2b
(79%). The result of screening various bases and additives
showed that installment of 2a on standard chloromethyl
derivatized Merrifield resin (∼2.2 mmol/g) is most effectively
accomplished by reaction of the enolate of 2a generated by
treatment of LDA in THF, see Table S1. This process can be
utilized to generate the Pyroacm linked resin 3a on a multigram
scale. The ATR-FTIR spectra of 3a (Figure S5) contains
intense and sharp peak at 1691 cm−1 corresponding to the
pyrrolidone carbonyl group. Introduction of the ester 2b to the
Merrifield resin was unsuccessful owing to its polymerization
during enolate formation. Immobilization of an appropriately
To evaluate the stability of Pyroacm resin, 6a1 (Scheme 3)
was subjected to acidic and basic conditions that are employed
in the Fmoc and Boc protocols. The results of ninhydrin tests
showed that 35% loss of peptide takes place after 24 h exposure
to 20 vol % piperidine (Table S3). In addition, the results of
Fmoc quantitative analysis showed that even exposure to
various acidic environments for long time periods does not
cause significant loss of the resin bound peptide (Table S4).
After demonstrating that the new Pyroacm resin can be
employed to synthesize model tripeptides, our attention next
B
DOI: 10.1021/acs.orglett.6b00115
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focused on the use of this resin to the preparation of the host-
defense skin peptides Riparin 1.2 and Riparin 1.1.11 For this
purpose, the appropriately blocked cysteine containing resin
5a1 was transformed to the resin linked undecapeptide 9a1
using Fmoc SPPS protocol (Scheme 4).
as an alternate sacrificial reaction site, 10 is generated in a
highly pure crude state (Table 1, entry 5). The second
procedure, method b (Table 2), can be employed to obtain the
Table 2. Cleavage Cocktail To Obtain Side Chain
a
Deprotected Disulfenyl Riparin 1.2 (11)
Scheme 4. Synthesis of Riparin 1.2
percentage
b
(%)
entry
1
reagent
11
67
11a yield of 11 (%)c
TFA:ScmCl
(99.75:0.25)
26
20
29
ND
ND
ND
47
2
3
4
5
6
TFA:H2O:ScmCl (94.75:5:0.25)
71
TFA:TIPS:ScmCl
(94.75:5:0.25)
65
TFA:PhOH:ScmCl
(94.75:5:0.25)
92.7
91.2
88
0.8
TFA:PhOH:H2O:ScmCl
(89.75:5:5:0.25)
1.2
2.1
46
TFA:PhOH:H2O:TIPS:ScmCl
(87.75:5:5:2:0.25)
09
a
Reactions carried out at 0.01 mmol scale. Product and byproducts in
b
the crude written in percentage. Yield based on purified product after
preparative HPLC. ND: not determined.
side chain deprotected target 11 directly from 9a1. However, we
observed that the treatment of 9a1 with a TFA:ScmCl mixture
in a ratio of 99.75:0.25 (i.e., ca. 4 equiv of ScmCl) gave rise to
11a (Scheme 4), containing Acm substitution at the aromatic
ring of tyrosine residue in 26% (Table 2, entry 1).12 Addition of
scavangers, such as 5 vol % TIPS or 5 vol % H2O, to the
reagent mixture was not helpful (29% and 20% of 11a,
respectively, in Table 2, entries 3 and 2). Finally, inclusion of 5
vol % phenol as a scavenger suppressed the production of 11a
to less than 1% and generated 11 in a highly pure crude state
(Table 2, entry 4). Surprisingly, the presence of H2O had no
effect on the yield of 11, but the presence of 2 vol % TIPS leads
to a considerable decrease in the overall yield (Table 2, entries
5 and 6) which indicates that ScmCl is quenched by TIPS.
Finally, method c, involves treatment with TFA:TIP-
S:PhOH:H2O (88:2:5:5), i.e., reagent B for 2 h prior to
addition of 4 equiv of ScmCl in AcOH. This process cleanly
forms the desired product 11 (Scheme 4). In addition, the
crude 11 in 5 vol % of DTT in H2O gave a crude product
mixture containing 12 and 13 in a ratio of 95:5. Subjection of
the mixture to HPLC purification and treatment of the
separated products with 10 vol % DMSO in H2O for 24 h
gave 13. The crude 13 was purified by HPLC to obtain Riparin
1.2 with high purity (Scheme 4).
Method a, devised in studies of the Riparin 1.2 synthesis, was
utilized in the preparation of Riparin 1.1 (Scheme S1) via both
the respective Trt and Acm cysteine blocked intermediates, 14a
and 14b. The cleavage condition using 4 equiv of ScmCl in
AcOH was employed to produce the desired product 15 with a
high purity in its crude state from both 14a and 14b. During
cleavage of resin 14a (Trt) using ScmCl in CH2Cl2, no N-Trt
transfer took place. Moreover, the use of the reagent
combination TFA:ScmCl (99.75:0.25, i.e., 4 equiv of ScmCl)
transformed 14b to 16 in the absence of forming the Acm
substituted peptide, an observation that is in agreement with
the tyrosine alkylation hypothesis (11a) proposed for the case
of Riparin 1.2. Finally, use of the optimized conditions
The first, method a (Table 1), is patterned after the
recommended process for conversion of an Acm to a Scm
Table 1. Method a To Obtain Side Chain Protected
Disulfenyl Riparin 1.2 (10)
a
percentage (%)
yield of 10
b
entry
reagent
10 10a 10b 10c
(%)
1
2
3
CH2Cl2
34
63
63
68
87
49
21
2
2
6
1
4
ND
11
CH2Cl2:AcOH (9:1)
AcOH
15
4
14
3
23
c
4
CH2Cl2:MeOH (95:5)
2
ND
41
5
AcOH: Boc-Gly-OH
(97:3)
3
<1
<1
a
All reactions carried out at 0.01 mmol scale. Product and byproducts
b
in the crude written in percentage. Yield based on purified product
c
after preparative HPLC. 21% methyl esterification of 10 observed.
ND: not determined.
group on a resin (ScmCl in CH2Cl2). Under these conditions,
9a1 is converted to a mixture containing a major amount of 10a
(Scheme 4), a N-Acm derivative of the target (Table 1, entry
1), but in the LCMS trace of crude reaction, N-Scm
substitution has not been observed (Figure S6). Although
formation of 10a in this process was minimized to <3% by
using AcOH as an additive or alternative solvent containing
ScmCl. But the use of this promoted formation of the Boc-
deprotected product 10b (Scheme 4) containing a free lysine
residue of 10 and the t-butyl deprotected product 10c (Scheme
4) containing a free tyrosine residue of 10 (Table 1, entries 2
and 3). Use of 5 vol % MeOH yielded 21% of methyl
esterification at C-terminal of 10 (Table 1, entry 4). Finally, by
including 3 vol % Boc-Gly-OH in the reagent mixture to serve
C
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Organic Letters
Letter
developed for 9a1 was found to promote successful cleavage of
both 14a and 14b to generate 15 and 16, respectively. Further,
5 vol % DTT in H2O reduced S-sulfenyl bond of 16 to obtain
17. The HPLC purified 17 containing 10% aq DMSO required
24−48 h for oxidation to give Riparin 1.1(Scheme S1). All
crude reactions HPLC and LCMS have been checked, and
purified products subjected to LC MS/MS analysis (see SI).
The new Pyroacm resin, described above, has some
noteworthy advantages. It has enabled the synthesis of various
C-terminal cysteine containing peptides with high purity. The
Pyroacm resin based procedure is compatible with both Fmoc
and Boc SPPS. Moreover, in this method the linked C-terminal
cysteine does not undergo β-elimination and racemization
during Boc protocol. The Pyroacm resin protocol uses
inexpensive, commercially available reagents and conditions
that are sufficiently mild to be carried out up to a gram scale.
During cleavage conditions, method a is applicable to all the
amino acid residues; however, methods b and c will give rise to
sulfenylation of unprotected Trp residues.13 Our processes
(methods b and c) are still applicable when Trp (CHO) is
employed; the CHO group can be cleaved by brief treatment
with NaOH after disulfide bond formation to give free Trp.14
However, further research is needed to apply the Pyroacm resin
in broad spectrum.
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ASSOCIATED CONTENT
* Supporting Information
■
S
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The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b00115.
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Experimental procedures and characterization data of all
reaction via NMR, IR, HPLC, LC/MS, LC MS/MS data
(PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: ydgong@dongguk.edu.
Notes
The authors declare no competing financial interest.
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(14) Akaji, K.; Tatsumi, T.; Yoshida, M.; Kimura, T.; Fujiwara, Y.;
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ACKNOWLEDGMENTS
■
This research was supported by the Bio & Medical Technology
Development Program of the National Research Foundation
(NRF) funded by the Ministry of Science, ICT & Future
Planning (No. 2014M3A9A9073847), and also financially
supported by the Ministry of Trade, Industry & Energy
(MOTIE), and the Korea Institute for Advancement of
Technology (KIAT) through the industrial infrastructure
program for fundamental technologies (Grant No. M0000338).
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DOI: 10.1021/acs.orglett.6b00115
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