4-Methoxybenzyloxymethyl group as an Nπ-protecting group for histidine to eliminate side-chain-induced racemization in the Fmoc strategy

Article abstract of DOI:10.1016/j.tetlet.2011.07.065

The 4-methoxybenzyloxymethyl (MBom) group was introduced at the N ^π-position of histidine, and its utility was examined under the conditions for peptide synthesis by Fmoc strategy. The N^π-MBom group proved to prevent the risk of race

Full text of DOI:10.1016/j.tetlet.2011.07.065

Tetrahedron Letters 52 (2011) 4947–4949
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p
4-Methoxybenzyloxymethyl group as an N -protecting group for histidine
to eliminate side-chain-induced racemization in the Fmoc strategy
Hajime Hibino a, Yuji Nishiuchi a,b,
⇑
a SAITO Research Center, Peptide Institute, Inc., 7-2-9 Saito-asagi, Ibaraki, Osaka 567-0085, Japan
b Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
a r t i c l e i n f o
a b s t r a c t
p
Article history:
The 4-methoxybenzyloxymethyl (MBom) group was introduced at the N -position of histidine, and its
p
Received 27 June 2011
Revised 11 July 2011
Accepted 15 July 2011
Available online 23 July 2011
utility was examined under the conditions for peptide synthesis by Fmoc strategy. The N -MBom group
proved to prevent the risk of racemization during incorporation of the His residue and to possess all of the
chemical properties required for Fmoc chemistry. The side reaction associated with formaldehyde gener-
p
ated from the N -MBom group upon acidolysis could be effectively prevented by performing the standard
TFA treatment in the presence of methoxyamineÁhydrochloride (MeONH2ÁHCl).
Keywords:
Histidine
Ó 2011 Elsevier Ltd. All rights reserved.
Protecting group
4-Methoxybenzyloxymethyl (MBom) group
Racemization
In peptide synthesis, side reactions sometimes occur when
there is no appropriate protection of the imidazole ring of histidine
(His), because the nucleophilic imidazole ring easily undergoes N-
acylation. This may give rise to undesired acyl transfer reactions. In
particular, His derivatives are known to be prone to racemization
try that can be easily purified by crystallization, we introduced the
4-methoxybenzyloxymethyl (MBom) group into the N -nitrogen of
the imidazole ring (Fig. 1).
p
The synthesis of Fmoc-His(
1.6 The reported procedure for the His(
derivatives was modified for the preparation of the His(p
ones, in which the N -amino function of the intermediates was
protected by the Boc group while that of the former by the Z group.
This modification facilitated purification by crystallization to sepa-
p
-MBom) (1) is illustrated in Scheme
-Bom) and His( -Bum)
-MBom)
p
p
on activating and coupling processes involving the
the imidazole ring.1 Therefore, the regioselective protection of
the -nitrogen should enable avoidance of racemization. For this
p-nitrogen of
a
p
p
purpose, the N -benzyloxymethyl (Bom) group has been widely
employed in Boc chemistry as it can effectively suppress the risk
of racemization and can be readily removed by HF or TfOH.2 An-
rate the undesired s-isomer from the product, thus making the
present synthetic procedure available for large-scale production
without any obstacles. Thus, regioselective alkylation using
4-methoxybenzyloxymethyl chloride (MBom-Cl) onto Boc-His
s
other group, the N -Trt group, is often used in combination with
a
N -Fmoc protection.3 Although the steric hindrance of the Trt
s
group may help reduce racemization to a certain extent, the N -
(s-Ac)-OMe (3) provided Boc-His(p-MBom)-OMe, which was
protecting group cannot inherently prevent racemization. To serve
subsequently saponified by NaOH aq to give Boc-His(p-MBom)
p
as the N -protecting group on His compatible with Fmoc chemis-
p
p
try, N -t-butoxymethylhistidine4 [His(Bum)] and N -1-adamantyl-
oxymethylhistidine5 [His(1-Adom)] have been developed. These
are ideal His derivatives for Fmoc chemistry in terms of offering
O
FmocHN CH C OH
CH2
p
N -protection and TFA lability. However, the preparation of these
protected derivatives is not straightforward and also requires col-
s
umn chromatography on silica gel to separate the N -isomer from
the desired one. This would not only hamper the purification pro-
cedure but also large-scale production, preventing such derivatives
from gaining widespread application in peptide synthesis. In order
O
N
N
MeO
p
to find an N -protected His derivative applicable for Fmoc chemis-
4-Methoxybenzyloxymethyl
(MBom)
⇑
Corresponding author. Tel.: +81 72 643 4411; fax: +81 72 643 4422.
E-mail address: yuji@peptide.co.jp (Y. Nishiuchi).
Figure 1. Structure of Fmoc-His(p-MBom) (1).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.07.065

4948
H. Hibino, Y. Nishiuchi / Tetrahedron Letters 52 (2011) 4947–4949
O
BocHN CH C OMe
CH2
O
O
BocHN CH C OH
CH2
BocHN CH C OMe
Ac2O
1) MBom-Cl
CH2
MBom
2) NaOH aq.
N
N
N
60% from 2
NH
N
N
Ac
2
3
4
O
H2N CH C OH
CH2
O
FmocHN CH C OH
CH2
Fmoc-OSu
95%
TMSOTf
MBom
MBom
N
2,6-lutidine
88%
N
N
N
5
Fmoc-His(π-MBom) (1)
Scheme 1. Synthesis of Fmoc-His(p-MBom) (1).
(4). Recrystallization of 4 could be effectively performed to remove
the contaminant, that is, the -isomer, without resorting to column
chromatography. To convert 4 into Fmoc-His(
Table 2
Racemization during synthesis of the model peptide, Z-Ala-His-Pro, as a function of
the His protecting group
s
a
p
-MBom) (1), the N -
Imidazole protection
-Trt
-MBom
Racemizationa (%)
Boc group on 4 was selectively cleaved by treatment with TMSOTf
p
in the presence of 2,6-lutidine7 with no loss of the N -MBom group
s
p
3.2
<0.2b
and the resulting
a-amino function was protected by the Fmoc
group. The optical purity of 1 synthesized in this manner was
>99.9% as determined by Marfey’s method after removing the pro-
tected groups.8,9
a
Defined as (Z-Ala-D-His-Pro)/(Z-Ala-His-Pro + Z-Ala-D-His-Pro) Â 100.
b
Below detection limit.
The stability and removability of the MBom group under the
standard conditions used for Fmoc-SPPS were examined by
with Fmoc (or Z)-amino acid/HBTU/DIEA (each of 1.5 equiv) in
DMF. As shown in Table 2, Fmoc-His( -MBom) (1) was found to
be accompanied by virtually no racemization on the activating
measuring the amount of recovered His(
The results are summarized in Table 1 in comparison with those
of the -Trt group. The MBom group was found to be completely
p-MBom) on RP-HPLC.
p
s
and coupling steps, while the regioisomeric derivative, that is,
stable during the repetitive Fmoc deprotection reaction using
20% piperidine/DMF but readily removable by TFA in the same
manner as the Trt group. On the other hand, the MBom group re-
mained attached to the imidazole ring during the detachment of
the protected peptides with a free carboxyl group from the Trt(2-
Cl) resin by treatment with DCM/TFE/AcOH (v/v, 3/1/1),10 while
the Trt group is known to be susceptible to this condition. The par-
tial loss of the Trt group on the His residue of the protected peptide
segments may not only hamper their purification procedure but
also be accompanied by side reactions involving the nucleophilic-
ity of the imidazole ring. These results demonstrated that the
MBom group for the His residue can offer permanent Nim-protec-
tion throughout the synthesis of the protected peptides. Next, the
Fmoc-His(
s
-MBom),12 led to considerable racemization (16%).
p
These results proved that the N -MBom group possessed all of
the chemical properties required for the Fmoc strategy.
When the His(p-Bom)- or His(p-Bum)-containing peptide is
treated with HF or TFA, respectively, formaldehyde is generated
from the respective Nim-protecting groups. This will lead to
hydroxymethylated modification of
a- and e-amino groups
although the extent is inconsequential. On the other hand, formal-
dehyde reacts almost quantitatively with a Cys residue located at
the N-terminus to produce a thiazolidyl (Thz)-peptide during isola-
tion from a HF or TFA cleavage mixture.13 To circumvent this con-
version, methoxyamine hydrochloride (MeONH2ÁHCl) has been
recommended as a formaldehyde scavenger in the acidolytic cleav-
p
suppressive effect of N -MBom on racemization during the incor-
age reaction.11,13 Here, we applied Fmoc-His(
p-MBom) to the syn-
poration of His was evaluated by synthesizing a model peptide,
Z-Ala-His-Pro.11 The peptide chain was elongated onto a Pro-
Trt(2-Cl) resin by the 1-min preactivation procedure of coupling
thesis of cystenyl-angiotensin II (Cys-Ang II: CDRVYIHPF)14 to
examine the by-product formation associated with the use of this
protecting group. As was expected, Thz formation arising from
the generation of formaldehyde during TFA treatment was almost
completely suppressed by the addition of MeONH2ÁHCl (5 equiv) to
the reaction mixture (Table 3). Besides formaldehyde, upon acidol-
ysis, the MBom group also generates an electrophilic alkylating
species, methoxybenzyl cation, that can cause alkylation of the
susceptible residues such as Cys and Trp. When cleaving the pep-
tide from a Wang resin, resin-bound carbocations lead to reattach-
ment of the initially cleaved peptide at Cys or Trp. The alkylation of
Trp is negligible when its indole ring is protected by the Boc group,
which is known to produce the Nin-carbamic acid compound to
Table 1
Recovery of His(X) under various conditions
Conditions
Recovery of His(X) (%)a
-MBom -Trt
X=
p
s
20% Piperidine/DMF
TFA/H2O (v/v, 95/5)
DCM/TFE/AcOH (v/v, 3/1/1)
rt, 2 h
rt, 1 h
rt, 8 h
100
0
100
100
0
64
a
Determined by HPLC (220 nm).

H. Hibino, Y. Nishiuchi / Tetrahedron Letters 52 (2011) 4947–4949
4949
Table 3
4. Colombo, R.; Colombo, F.; Jones, J. H. J. Chem. Soc., Chem. Commun. 1984, 292–
293.
Effect of additives on Cys modification during TFA deprotection
5. Okada, Y.; Wang, J.; Yamamoto, T.; Mu, Y.; Yokoi, T. J. Chem. Soc., Perkin Trans. 1
1996, 17, 2139–2143.
Additivea
Ratio of Cys-Ang II to Cys(X)-Ang II
6. All of the amino acid derivatives used in the present study possess
configuration.
7. Bastiaans, H. M. M.; Harry, J. L. V. D. B.; Ottenheijm, C. J. J. Org. Chem. 1997, 62,
3880–3889.
L-
Cys-Ang
II
Thz-Ang
II
Cys(MeOBzl)-Ang
II
None
88
91
99
5
<1
<1
7
8
<1
8. Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
MeONH2ÁHCl (5 equiv)
9. Fmoc-His(p
-MBom) (1): 1H NMR (DMSO-d6) 2.90–3.17 (m, 2H), 3.71 (s, 3H),
MeONH2ÁHCl
(5 equiv) + PhSHb
4.15–4.38 (m, 6H), 5.38 (q, 2 H, J = 10.0 Hz) 6.74–6.87 (m, 3H), 7.15–7.42 (m,
6H) 7.60–7.94 (m, 6H); 13C NMR (DMSO-d6) 25.2, 46.6, 50.1, 53.2, 55.0, 63.8,
65.7, 69.0, 73.2, 113.7, 119.8, 120.1, 125.2, 126.8, 127.0, 127.1, 127.6, 127.8,
128.9, 129.5, 138.3, 140.6, 143.7, 145.2, 155.9, 158.8, 172.9; ESI MS: Calcd for
a
The protected peptides were treated with TFA/TIS/H2O (v/v, 95/2.5/2.5) in the
presence of the additives listed in Table 3 at rt for 1 h.
[C30H29N3O6+H]+ 528.21, found 528.2; [
a
]
D
À8.3 (c 1.0, DMF); Elemental Anal.
b
PhSH was used in the same volume as that of H2O.
Calcd for C30H29N3O6; C, 68.30; H, 5.54; N, 7.96. Found C, 68.21; H, 5.58; N,
7.92; rt: 16.2 min (HPLC conditions: column, YMC-ODS AA12S05-1546WT at
40 °C; eluent, 20–70% MeCN/0.1% TFA (25 min); flow rate, 1 mL/min; detection,
220 nm.)
prevent such electrophilic additions during the final cleavage.15 To
avoid that of Cys, however, performing TFA cleavage in the pres-
ence of thiols is necessary. This measure using thiols could effec-
tively prevent alkylation of Cys with methoxybenzyl cation
generated from the MBom group.
10. Barlos, K.; Gatos, D. Pept. Sci. 1999, 51, 266–278.
11. Mergler, M.; Dick, F.; Sax, B.; Schwindling, J.; Vorherr, T. J. Pept. Sci. 2001, 7,
502–510.
12. By employing the same procedure as that for 1, Fmoc-His(
prepared from Boc-His( -MBom)-OMe obtained by alkylating Boc-His-OMe (2)
with MBom-Cl. Fmoc-His(
-MBom): 1H NMR (DMSO-d6) 2.90–3.16 (m, 2H),
s-MBom) was
s
In summary, the MBom group was shown to be a suitable pro-
s
tecting group for Fmoc chemistry to protect the
p
-nitrogen on the
3.71 (s, 3H), 4.13–4.37 (m, 6H), 5.36 (q, 2H, J = 10.0 Hz) 6.75–6.87 (m, 3H),
7.15–7.41 (m, 6H) 7.60–7.94 (m, 6H); 13C NMR (DMSO-d6) 25.1, 46.6, 50.1,
53.2, 55.2, 63.8, 65.7, 69.0, 73.2, 113.6, 119.8, 120.1, 125.2, 126.8, 127.0, 127.1,
127.7, 127.8, 128.9, 129.5, 138.3, 140.6, 143.7, 145.2, 155.8, 158.8, 172.7;
Elemental Anal. Calcd for C30H29N3O6; C, 68.30; H, 5.54; N, 7.96. Found: C,
68.31; H, 5.62; N, 7.78; rt: 16.5 min (The same HPLC conditions described in
Ref. 9).
His residue in order to eliminate the side-chain-induced racemiza-
tion. In particular, the MBom group proved to be indispensable for
preparing the protected His-containing peptide segments on acid-
sensitive linkers for the convergent synthesis.
13. Taichi, M.; Kimura, T.; Nishiuchi, Y. Int. J. Pept. Res. Ther. 2009, 15, 247–
253.
References and notes
14. Protected Cys-Ang II was assembled with an ABI 433A (Forester, CA, USA) using
Fmoc strategy on Wang resin. The peptide chain was elongated using the
FastMocÒ protocol of coupling with Fmoc-amino acid/HCTU/6-Cl-HOBt/DIEA
(4/4/4/8 equiv) in NMP. The following side-chain-protected amino acids were
employed: Cys(Trt), Asp(OtBu), His(MBom), Arg(Pbf) and Tyr(tBu).
15. Fields, C. G.; Fields, G. B. Tetrahedron Lett. 1993, 34, 6661–6664.
1. Isidro-Llobet, A.; Álvarez, M.; Albericio, F. Chem. Rev. 2009, 109, 2455–2504.
2. (a) Jones, J. H.; Ramage, W. I. J. Chem. Soc., Chem. Commun. 1978, 472–473; (b)
Jones, J. H.; Stachulski, A. V. J. Chem. Soc., Perkin Trans. 1 1979, 2261–2267.
3. Barlos, K.; Papaioannou, D.; Theodoropoulos, D. J. Org. Chem. 1982, 47, 1324–
1326.