4-methoxybenzyloxymethyl group, a racemization-resistant protecting group for cysteine in fmoc solid phase peptide synthesis

Article abstract of DOI:10.1021/ol300592w

The 4-methoxybenzyloxymethyl (MBom) group was introduced for sulfhydryl protection of Cys in combination with Fmoc chemistry. The MBom group proved to substantially suppress racemization of Cys during its incorporation mediated by phosphonium or uronium reagents. Furthermore, this group was found to significantly reduce racemization of the C-terminal Cys linked to a hydroxyl resin during repetitive base treatment, in comparison with the usually used trityl (Trt) and acetamidomethyl (Acm) groups.

Full text of DOI:10.1021/ol300592w

ORGANIC
LETTERS
2012
Vol. 14, No. 7
1926–1929
4-Methoxybenzyloxymethyl Group,
a Racemization-Resistant Protecting
Group for Cysteine in Fmoc Solid
Phase Peptide Synthesis
Hajime Hibino† and Yuji Nishiuchi*,†,‡
SAITO Research Center, Peptide Institute, Inc., Ibaraki, Osaka 567-0085, Japan, and
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka,
Osaka 560-0043, Japan
yuji@peptide.co.jp
Received March 8, 2012
ABSTRACT
The 4-methoxybenzyloxymethyl (MBom) group was introduced for sulfhydryl protection of Cys in combination with Fmoc chemistry. The MBom
group proved to substantially suppress racemization of Cys during its incorporation mediated by phosphonium or uronium reagents.
Furthermore, this group was found to significantly reduce racemization of the C-terminal Cys linked to a hydroxyl resin during repetitive base
treatment, in comparison with the usually used trityl (Trt) and acetamidomethyl (Acm) groups.
Disulfide linkages play an important role in the forma-
tion and stabilization of distinct three-dimensional struc-
tures in naturally occurring peptides and proteins. For the
synthesis of such Cys-rich molecules, native chemical
ligation (NCL)1 involving the coupling of a peptide thio
ester and a cysteinyl-peptide is an advantageous approach.
In the course of peptide assembly, protection of the
sulfhydryl group on Cys is indispensable for avoiding side
reactions related to its high nucleophilicity, such as acyla-
tion, alkylation, or oxidation. For this purpose, many side-
chain protecting groups for Cys have been developed.2
Among them, the trityl (Trt) and acetamidomethyl (Acm)
groups are widely accepted as a protecting group for Cys in
the Fmoc strategy. During incorporation of these Cys
derivatives onto the growing peptide chain, however,
considerable base-catalyzed racemization of the Cys resi-
due is known to always occur at the activating and
coupling steps with phosphonium or uronium reagents
such as PyBOP or HBTU, respectively.3 This may hamper
purification of the products including those obtained after
the NCL and disulfide formation reactions. Therefore, the
carbodiimide-mediated coupling method has been recom-
mended to reduce the racemization rate during Cys in-
corporation to acceptable levels (<1.0%) although this
method has no advantage in terms of coupling efficiency
over that using phosphonium or uronium reagents.4 The
synthetic procedure would be complicated if the protocols
for coupling reagents had to be changed every time in-
corporation of Cys is performed, especially in the case of
† Peptide Institute, Inc.
‡ Osaka University.
(1) (a) Dawson, P.; Muir, T.; Clark-Lewis, I.; Kent, S. Science 1994,
266, 776. (b) Dawson, P. E.; Kent, S. B. H. Annu. Rev. Biochem. 2000, 69,
923. (c) Hackenberger, C. P. R; Schwarzer, D. Angew. Chem., Int. Ed.
2008, 47, 10030.
(3) Kaiser, E. T.; Nicholson, G. J.; Kohlbau, H. J.; Voelter, W.
Tetrahedron Lett. 1996, 37, 1187.
(4) Incorporation of Cys was performed by a protocol of coupling
with Fmoc-amino acid/N,N0-diisopropylcarbodiimide (DIPCDI)/
1-hydroxybenzotriazole (HOBt) (4:4:4 equiv with respect to the peptide;
5-min preactivation) in DMF. See: (a) Angell, Y. M.; Alsina, J.;
Albericio, F.; Barany, G. J. Pept. Res. 2002, 60, 292–299. (b) Han, Y.;
Albercio, F.; Barany, G. J. Org. Chem. 1997, 62, 4307.
ꢀ
(2) (a) Isidro-Llobet, A.; Alvarez, M.; Albericio, F. Chem. Rev. 2009,
109, 2455. (b) Otaka, A.; Morimoto, H.; Fujii, N.; Koide, T.; Funakoshi,
S.; Yajima, H. Chem. Pharm. Bull. 1989, 37, 526. (c) Garcia, O.; Bofill, J.;
Nicolas, E.; Albercio, F. Eur. J. Org. Chem. 2010, 3631. (d) Shen, F.;
Zhang, Z.; Li, J.; Lin, Y.; Liu, L. Org. Lett. 2011, 13, 568.
r
10.1021/ol300592w
Published on Web 03/27/2012
2012 American Chemical Society

machine-assisted solid phase peptide synthesis (SPPS). In
addition to this, racemization of the C-terminal Cys ester-
ified to resins also occurs during the repetitive NR-Fmoc
deprotection using piperidine.5 Even when employing a
Trt-type resin with the aid of its steric hindrance, racemiza-
tion of the C-terminal Cys associated with the base treat-
ment is likely to occur.6 In order to find an S-protected Cys
derivative applicable for Fmoc chemistry that can effi-
ciently suppress racemization of Cys both when its incor-
poration is conducted with phosphonium or uronium
reagents and when it is linked to a resin via an ester linkage,
we introduced the 4-methoxybenzyloxymethyl (MBom)
group into the sulfhydryl function of Cys (Figure 1).
one is that in which formaldehyde reacts almost quantita-
tively with a Cys residue located at the N-terminus to
produce a thiazolidyl (Thz)-peptide during isolation from
an acidolysis mixture.9 This may hamper the preparation
of peptides having an N-terminal Cys residue used for the
subsequent NCL. To circumvent this conversion, methoxy-
amine hydrochloride (MeONH2 HCl) has been recom-
3
mended as a formaldehyde scavenger in the acidolytic
cleavage reaction.10 As for the methoxybenzyl cation, it
was reported that alkylation of susceptible residues such
as Cys can be effectively prevented by performing TFA
cleavage in the presence of thiol compounds.11 Thus, we
applied Fmoc-Cys(MBom) to the synthesis of cysteinyl-
angiotensin II (Cys-Ang II: CDRVYIHPF) to examine the
byproduct formation associated with the use of this pro-
tecting group. As expected, performing TFA cleavage using
the reagent K in the presence of MeONH2 HCl (5 equiv)
3
was found to be accompanied by virtually no side products
arising from formaldehyde and the methoxybenzyl cation
(Table 1).
Table 1. Effect of Additives on Cys Modification during TFA
Deprotection Using Reagent Ka
Figure 1. Structure of Fmoc-Cys(MBom) (1).
ratio of Cys-Ang II to Cys(X)-Ang II
The synthesis of Fmoc-Cys(MBom) (1) is illustrated in
Scheme 1. Fmoc-Cys-OAllyl (2)7 was alkylated using
4-methoxybenzyloxymethyl chloride (MBom-Cl) to pro-
vide Fmoc-Cys(MBom)-OAllyl (3), which was subse-
quently deallylated by treatment with Pd(PPh3)4 and
dimedone to give 1.
additive
none
Cys-Ang II Thz-Ang II Cys(MeOBzl)-Ang II
82
98
24
<1
<1
<1
MeONH2 HCl
3
(5 equiv)
a Reagent K: TFA/H2O/phenol/thioanisole/1,2-ethanedithiol (v/v,
82.5/5/5/5/2.5).
Scheme 1. Synthesis of Fmoc-Cys(MBom) (1)
Next, the suppressive effect of S-MBom on racemization
during incorporation of Cys was evaluated by synthesizing
a model peptide, Gly-Cys-Phe-NH2.4 The peptide chain
was elongated onto a Rink amide resin using the 1-min
preactivation procedure of coupling with Fmoc-amino
acid/HCTU/6-Cl-HOBt/DIEA (4/4/4/8 equiv) in DMF.
The results are summarized in Table 2 in comparison with
those of the Acm and Trt groups. Fmoc-Cys(MBom) was
found to be accompanied by an acceptable level of race-
mization (0.4%) on the activating and coupling steps in the
conventional SPPS compared with Fmoc-Cys(Trt) and
Fmoc-Cys(Acm) (8.0% and 4.8%, respectively). Even in
the case of the microwave-assisted SPPS performed at
The MBom group was found to be completely stable
during the repetitive Fmoc deprotection reaction using
20% piperidine/DMF but readily removable by TFA in
the same manner as the Trt group, although the MBom
group generates formaldehyde and an electrophilic
alkylating species, methoxybenzyl cation, upon TFA
cleavage.8 Formaldehyde can lead to hydroxymethylation
of the functional groups on peptides. A particularly serious
(9) (a) Mitchell, M. A.; Runge, T. A.; Mathews, W. R.; Ichhpurani,
A. K.; Harn, N. K.; Dobrowolski, P. J.; Eckenrode, F. M. Int. J. Pept.
Protein Res. 1990, 36, 350. (b) Yoshizawa-Kumagaye, K.; Inui, T.;
Nakajima, K.; Kimura, T.; Sakakibara, S. Pept. Res. 1991, 4, 84. (c)
Yoshizawa-Kumagaye, K.; Ishizu, T.; Isaka, S.; Tamura, M.; Ohkihara,
R.; Nishiuchi, Y.; Kimura, T. Protein Pept. Lett. 2005, 12, 579.
(10) (a) Taichi, M.; Kimura, T.; Nishiuchi, Y. Int. J. Pept. Res. Ther.
2009, 15, 247. (b) Mergler, M.; Dick, F.; Sax, B.; Schwindling, J.;
Vorherr, T. J. Pept. Sci. 2001, 7, 502.
(11) Trp is also susceptible to the alkylation with a methoxybenzyl
cation. In practical peptide synthesis, however, Trp alkylation is negli-
gible as long as its indole ring is protected by the Boc group, which can
produce the Nin-carbamic acid compound to prevent such electrophilic
attacks during TFA treatment. See: Fields, C. G.; Fields, G. B. Tetra-
hedron Lett. 1993, 34, 6661.
(5) Atherton, E.; Hardy, P.; Harris, D.; Matthews, B. Racemization
of C-terminal cysteine during peptide assembly. In Peptides 1990,
Proceeding of the Twenty-First European Peptide Symposium 1990; Giralt,
E., Andreu, D., Eds.; ESCOM Science Publishers B.V.: 1991; p 243.
(6) Fujiwara, Y.; Akaji, K.; Kiso, Y. Chem. Pharm. Bull. 1994, 42,
724.
(7) Branderhorst, H.; Liskamp, R.; Pieters, R. Tetrahedron 2007, 63,
4290.
(8) Hibino, H.; Nishiuchi, Y. Tetrahedron Lett. 2011, 52, 4947.
Org. Lett., Vol. 14, No. 7, 2012
1927

50 °C/80 °C,12 Fmoc-Cys(MBom) caused a significant
reduction in the level of racemization (0.8/1.3%) while
Fmoc-Cys(Trt) and Fmoc-Cys(Acm) led to a considerable
level of racemization (10.9/26.6% and 8.8/15.3%,
respectively) (Figure 2).
Table 2. Racemization during Synthesis of the Model Peptide,
Gly-Cys-Phe-NH2, as a Function of the Cys Protecting Groupa
racemization (%)b
microwave
microwave
X
conventional
50 °C
80 °C
Trt
8.0
4.8
0.4
10.9
8.8
26.6
15.3
1.3
Figure 3. Racemization of the C-terminal Cys linked to Trt resin
during exposure to 20% piperidine/DMF. Defined as (Bz-Ser-D-
Cys)/(Bz-Ser-L-Cys) ꢁ 100. More details are described in the
Supporting Information.
Acm
MBom
0.8
a The coupling reactions were performed using a 1-min preactivation
procedure of coupling with Fmoc-amino acid/HCTU/6-Cl-HOBt/
DIEA (4/4/4/8 equiv) in DMF. b Defined as (Gly-D-Cys-Phe-NH2)/
(Gly-L-Cys-Phe-NH2) ꢁ 100. More details can be found in the Supporting
Information.
Cys(Trt) and Cys(Acm) contained significant amounts of
isomers in that order. However, a much lower rate of
racemization (6.4%) was detected even after a 6-h treat-
ment with 20% piperidine/DMF when Cys(MBom) was
used (Figure 3). This indicated that more attention should
be paid to the suppression of racemization with the
C-terminal Cys linked to a hydroxyl resin, especially when
synthesizing a long peptide. These results suggested that
use of the MBom group effectively suppresses racemiza-
tion at the C-terminal Cys caused by the base treatment,
probably due to an electron-donating effect. Racemization
occurs through oxazolone and direct enolization. In parti-
cular, Cys tends to be racemized via enolization due
to stabilization of the carbanion formed on R-proton
abstraction.13 To destabilize this enol form, therefore, an
S-protecting group that possesses an electron-donating
effect would be essential to prevent racemization of Cys.14
In order to demonstrate the usefulness of the MBom
group on Cys, especially in the NCL chemistry, we synthe-
sized human glucagon-like peptide 1 (GLP-1, 7ꢀ36 amide)15
by Ala ligation involving a ligationꢀdesulfurization
approach16 that consisted of NCL between the thioester
peptide (7ꢀ24) and Cys25-(26ꢀ36)-NH2 followed by de-
sulfurization to convert Cys25 to Ala25. When synthesizing
Cys25-(26ꢀ36)-NH2 by using Fmoc-Cys(Trt) with the aid
of HCTU/6-Cl-HOBt/DIEA, the product was found to be
contaminated by 7% of D-Cys25-(26ꢀ36)-NH2 at a mod-
erate estimate. The diastereoisomer was eluted at the same
retention time as the desired peptide on RP-HPLC using
various gradient systems although the isocratic system
made it difficult to separate because it was a shoulder peak
Figure 2. HPLC profiles of the products, Gly-Cys-Phe-NH2,
obtained by performing MW-assisted SPPS at 80 °C. Incorpora-
tion of Cys was performed by coupling Fmoc-Cys(Trt) (a) and
Fmoc-Cys(MBom) (b). HPLC conditions are described in the
Supporting Information.
As for racemization of the C-terminal Cys esterified to a
Trt-type resin, the Bz-Ser(tBu)-Cys(X)-NovaSynTGT re-
sin, where X is MBom, Acm, or Trt, was exposed to 20%
piperidine/DMF for a given period of time to estimate
the racemization rate arising during the repetitive Fmoc
deprotection reactions.6 The products prepared using
(13) Barber, M.; Jones, J.; Witty, M. J. Chem. Soc., Perkin Trans. 1
1979, 10, 2425.
(14) Williams, S.; Albercio, F. Giralt, E. Chemical Approaches to the
Synthesis of Peptides and Proteins; CRC Press: New York, 1997.
(15) Turton, M.; O’Shea, D.; Gunn, I.; Beak, S.; Edwards, C.;
Meeran, K.; Chol, S.; Taylor, G.; Heath, M.; Lambert, P.; Wilding, J.;
Smith, D.; Ghatel, M.; Herbert, J.; Bloom, S. Nature 1996, 361, 362.
(16) (a) Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123, 526.
(b) Wan, Q.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2007, 46, 9248. (c)
Haase, C.; Rohde, H.; Seitz, O. Angew. Chem., Int. Ed. 2008, 47, 6807.
(d) Rohde, H.; Seitz, O. Peptide Sci. 2010, 94, 551.
(12) Palasek, S. A.; Cox, Z. J.; Collins, J. M. J. Pept. Sci. 2007, 13,
143.
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Org. Lett., Vol. 14, No. 7, 2012

respectively, could not be separated from the respective
desired products by RP- and IEX-HPLC. Thus, there were
no practical purification procedures available for remov-
ing these side products. In general, a diastereoisomer with
racemization somewhere in its sequence tends to be diffi-
cult to separate from the intact peptide as its chain
length increases. Therefore, it is important to exclude the
risk of racemization with incorporation of Cys by using
Fmoc-Cys(MBom) during the course of the chain assem-
bly. In fact, racemization with the N-terminal Cys in
Cys25-(26ꢀ36)-NH2 was found to be negligible when using
Fmoc-Cys(MBom) as shown in Figure 4. This measure
using Fmoc-Cys(MBom) can facilitate the avoidance of
ambiguities in the quality of the synthesized peptides.
In summary, the MBom group on Cys was demon-
strated to possess all of the chemical properties required
for the Fmoc strategy and to effectively prevent racemiza-
tion of Cys during its incorporation mediated even by
phosphonium or uronium reagents as well as that of the
C-terminal Cys esterified to the solid support during
repetitive base treatment.
Figure 4. HPLC profiles of Cys25-[GLP-1(26ꢀ36)-NH2]. (a) A
mixture of L-Cys25- and D-Cys25-[GLP-1(26ꢀ36)-NH2]. The
product obtained by using Fmoc-Cys(Trt) (b) or Fmoc-Cys-
(MBom) (c). HPLC conditions are described in the Supporting
Information.
Supporting Information Available. Experimental pro-
cedures, NMR charts for key compounds. This material
is available free of charge via the Internet at http://pubs.
acs.org.
(Figure 4). Even after NCL and desulfurization, the
racemized peptides arising from D-Cys25 and D-Ala25,
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 7, 2012
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