Synthesis and application of Nα-Fmoc-Nπ-4-methoxybenzyloxymethylhistidine in solid phase peptide synthesis

Article abstract of DOI:10.1002/psc.2464

The 4-methoxybenzyloxymethyl (MBom) group was introduced at the N^π-position of the histidine (His) residue by using a regioselective procedure, and its utility was examined under standard conditions used for the conventional and the microwave (MW)-assisted solid phase peptide synthesis (SPPS) with 9-fluorenylmethyoxycarbonyl (Fmoc) chemistry. The N^π-MBom group fulfilling the requirements for the Fmoc strategy was found to prevent side-chain-induced racemization during incorporation of the His residue even in the case of MW-assisted SPPS performed at a high temperature. In particular, the MBom group proved to be a suitable protecting group for the convergent synthesis because it remains attached to the imidazole ring during detachment of the protected His-containing peptide segments from acid-sensitive linkers by treatment with a weak acid such as 1% trifluoroacetic acid in dichloromethane. We also demonstrated the facile synthesis of Fmoc-His(π-MBom)-OH with the aid of purification procedure by crystallization to effectively remove the undesired τ-isomer without resorting to silica gel column chromatography. This means that the present synthetic procedure can be used for large-scale production without any obstacles.

Full text of DOI:10.1002/psc.2464

Research Article
Received: 8 September 2012
Revised: 8 October 2012
Accepted: 9 October 2012
Published online in Wiley Online Library: 30 October 2012
(wileyonlinelibrary.com) DOI 10.1002/psc.2464
Synthesis and application of Na-Fmoc-Np-4-
methoxybenzyloxymethylhistidine in solid
phase peptide synthesis
Hajime Hibino,a Yasuyoshi Mikib and Yuji Nishiuchia,c*
The 4-methoxybenzyloxymethyl (MBom) group was introduced at the Np-position of the histidine (His) residue by using
a regioselective procedure, and its utility was examined under standard conditions used for the conventional and the
microwave (MW)-assisted solid phase peptide synthesis (SPPS) with 9-fluorenylmethyoxycarbonyl (Fmoc) chemistry. The
Np-MBom group fulfilling the requirements for the Fmoc strategy was found to prevent side-chain-induced racemization
during incorporation of the His residue even in the case of MW-assisted SPPS performed at a high temperature. In particular,
the MBom group proved to be a suitable protecting group for the convergent synthesis because it remains attached to the
imidazole ring during detachment of the protected His-containing peptide segments from acid-sensitive linkers by treatment
with a weak acid such as 1% trifluoroacetic acid in dichloromethane. We also demonstrated the facile synthesis of Fmoc-His
(p-MBom)-OH with the aid of purification procedure by crystallization to effectively remove the undesired t-isomer without
resorting to silica gel column chromatography. This means that the present synthetic procedure can be used for large-scale
production without any obstacles. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: histidine; 4-methoxybenzyloxymethyl (MBom) group; microwave (MW)-assisted solid phase peptide synthesis (SPPS);
protecting group; racemization
This would hamper large-scale production, thus preventing such
Introduction
derivatives from gaining widespread application in the practical
peptide synthesis. To obtain an Np-protected His derivative
In the synthesis of histidine (His)-containing peptides, appropriate
protection of the imidazole ring of the His residue is indispensable
for avoiding side reactions associated with the nucleophilicity of
the imidazole ring, such as Nim-acylation followed by acyl transfer
reactions, during the course of chain assembly. In particular, the
His residue is known to be extremely prone to racemization in
activating and coupling processes involving the p-nitrogen of
the imidazole ring [1]. Therefore, regioselective protection of the
p-nitrogen should enable avoidance of racemization during incor-
poration of His derivatives onto the growing peptide chains [2]. In
Boc chemistry, the Np-benzyloxymethyl (Bom) group is widely
accepted as a protecting group for His because it can effectively
suppress the risk of racemization and can be readily removed by
HF or trifluoromethanesulfonic acid [3]. On the other hand, the
Nt-Trt group is often used in combination with Na-Fmoc protec-
tion [4]. Although the steric hindrance and electron withdrawing
effect of the Trt group on the t-nitrogen may help reduce racemi-
zation to a certain extent, the Nt-protecting group cannot
inherently prevent racemization. In particular cases such as the
formation of ester bonds or when the amino component is
sterically hindered, the Nt-Trt group on His raises the risk of
racemization. In addition, MW-assisted SPPS performed using
Fmoc-His(t-Trt)-OH results in a serious level of racemization [5].
To offer the Np-protection of His compatible with Fmoc chemistry,
the Np-t-butoxymethyl and Np-1-adamantyloxymethyl groups
have been developed [6,7]. However, the preparation of Fmoc-
His-OH derivatives substituted with these protecting groups at
the p-nitrogen requires a purification procedure using column
chromatography on silica gel to separate the undesired t-isomer.
applicable to Fmoc chemistry and easily purified by crystallization,
we introduced the MBom [8], 2,4-DMBom, and 3,4-DMBom
groups at the p-nitrogen of the imidazole ring. Among them,
the MBom group was found to be a suitable protecting group of
the His residue applicable for Fmoc chemistry to eliminate the
side-chain-induced racemization during incorporation of His
performed even by MW-assisted SPPS at a high temperature.
*
Correspondence to: Yuji Nishiuchi, SAITO Research Center, Peptide Institute, Inc.,
7-2-9 Saito-Asagi, Ibaraki-shi, Osaka 567-0085, Japan. E-mail: yuji@peptide.co.jp
a SAITO Research Center, Peptide Institute, Inc., Ibaraki, Osaka 567-0085, Japan
b School of Pharmaceutical Sciences, Kinki University, Higashi-Osaka, Osaka
577-8502, Japan
c
Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
Abbreviations used: Ang II, angiotensin II; Boc, t-butoxycarbonyl; tBu, t-butyl;
6-Cl-HOBt, 6-chloro-1-hydroxybenzotriazole; DCC, N,N0-dicyclohexylcarbodii-
mide; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DMAP,
4-dimethylaminopyridine; 2,4-DMBom, 2,4-dimethoxybenzyloxymethyl; 3,4-
DMBom, 3,4-dimethoxybenzyloxymethyl; ESI MS, electrospray ionization mass
spectrometry; Fmoc, 9-fluorenylmethyoxycarbonyl; HCTU, 1-[bis(dimethylamino)
methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate; HPLC,
high performance liquid chromatography; MBom, 4-methoxybenzyloxymethyl;
MW, microwave; NPW30, neuropeptide W-30; Pbf, 2,2,4,6,7-pentamethyldihy-
drobenzofuran-5-sulfonyl; RP-HPLC, reversed phase HPLC; SPPS, solid-phase
peptide synthesis; TFA, trifluoroacetic acid; THCar, tetrahydroxy-b-carboline;
Thz, thiazolidine; TMSOTf, trimethylsilyl triflate; Trt, trityl; Trt(2-Cl), 2-chlorotrityl;
Z, benzyloxycarbonyl.
J. Pept. Sci. 2012; 18: 763–769
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

HIBINO, MIKI AND NISHIUCHI
172.9.; ESI MS Calcd for [C30H29N3O6 + H]+ 528.21, found 528.2;
[a]D ꢂ20.0 (c 1.12, DMF).
Materials and Methods
All reagents and solvents were obtained from the Peptide Institute,
Inc. (Osaka, Japan), Wako Chemical (Osaka, Japan), Tokyo Chemical
Industry (Tokyo, Japan), and Watanabe Chemical Industries
(Hiroshima, Japan). Analytical HPLC was performed on a Shimadzu
liquid chromatograph Model LC-10AT (Kyoto, Japan) with a DAISO-
PAK SP-120-5-ODS-BIO (4.6 ꢀ 150 mm) using a flow rate of 1 ml/min
and the following solvent systems: 0.1% TFA in H2O (A) and 0.1%
TFA in MeCN (B). Purities are based upon area percent of the peaks
detected at 220 nm. High resolution mass spectra were measured
with a Bruker Esquire 200T (Billerica, MA, USA). Molecular weights
were measured with a MALDI-TOF MS (Voyager-DESTR, Applied
Fmoc-(2,4-DMBom)-OH (1b) and Fmoc-(3,4-DMBom)-OH (1c)
were prepared as described earlier for Fmoc-His(p-MBom)-OH
(1a) by using 3,4-DMBom-Cl and 2,4-DMBom-Cl, respectively.
Fmoc-His(2,4-DMBom)-OH (1b); 1H NMR (d6-DMSO) d = 7.89
(2H, d, J = 6.87 Hz), 7.75 (1H, d, J = 8.70 Hz), 7.66 (2H, d,
J = 7.79 Hz), 7.48 (2H, t, J = 7.20 Hz), 7.30 (2H, t, J = 7.20 Hz), 7.26
(2H, d, J = 8.80 Hz), 6.65 (1H, d, J = 2.70 Hz), 6.50 (1H, d, J = 8.80,
2.70 Hz), 4.73 (2H, s), 4.44 (2H, dd, J = 16.03, 10.99 Hz), 4.35–4.10
(4H, m), 3.71 (3H, s), 3.10–2.78 (2H, m).; 13C NMR (d6-DMSO)
d = 32.0, 46.6, 54.3, 55.0, 55.8, 65.8, 66.7, 72.7, 100.3, 106.2,
118.2, 120.1, 125.2, 125.3, 127.1, 127.6, 128.9, 137.3, 140.7, 143.8,
156.0, 158.8, 159.8, 172.3; HRMS Calcd for [C31H31N3O7H]+, m/z
558.2235, found 558.2238.
Biosystems) and an ESI MS (HP1100 LC/MSD). H and 13C NMR
1
spectra were recorded on a JEOL-ECX400 spectrometer (Tokyo,
Japan) in dimethyl sulfoxide-d6 (d6-DMSO) with the solvent residual
peak (d6-DMSO: 1H = 2.49 ppm, 13C = 39.52 ppm) as an internal
reference unless otherwise stated.
Fmoc-His(3,4-DMBom)-OH (1c); 1H NMR (d6-DMSO) d = 7.88
(2H, d, J = 6.87 Hz), 7.75 (1H, d, J = 8.70 Hz), 7.70 (2H, d, J = 7.79Hz),
7.40 (2H, t, J = 7.20Hz), 7.30 (2H, t, J = 7.20 Hz), 7.10 (2H, d,
J = 2.70 Hz), 6.87 (1H, d, J = 8.80, 2.70 Hz), 6.58 (1H, d, J = 8.80Hz),
4.73 (2H, s), 4.44 (2H, dd, J= 16.03, 10.99 Hz), 4.35–4.10 (4H, m),
3.71 (3H, s), 3.10–2.78 (2H, m); 13C NMR (d6-DMSO) d = 32.0, 46.6,
54.3, 55.0, 55.2, 65.8, 68.7, 72.7, 112.7, 114.6, 120.1, 121.9, 125.2,
125.3, 127.1, 127.6, 128.2, 128.9, 137.3, 140.7, 143.8, 148.8, 156.0,
158.8, 172.3; HRMS: Calcd for [C31H31N3O7H]+: m/z 558.2235, found
558.2237.
Synthesis of Fmoc-His(p-MBom)-OH (1a) and its Related
Compounds (1b, 1c)
Boc-His(p-MBom)-OH (4a)
A solution of Boc-His(t-Ac)-OMe (3a) (6.10 g, 19.6 mmol) and
4-methoxybenzyloxymethylchloride (MBom-Cl: 4.51g, 24.5mmol)
in CH2Cl2 (60ml) was stirred at room temperature for 4 h. After
removal of the solvent, the residue was triturated with diethyl ether
to give Boc-His(p-MBom)-OMe (HCl form, 10.6 g). A solution of
Boc-His(MBom)-OMe obtained previously in MeOH (40 ml) was
treated with 1 M NaOH aq. (40 ml) and stirred at room temperature
for 4 h. The reaction mixture was adjusted with 1 M HCl to pH 4.
After removal of the solvent, the residue was extracted with CHCl3.
The extract was washed with water and dried over Na2SO4. After
removal of the solvent, the residue was triturated with AcOEt to
give Boc-His(p-MBom)-OH (4.92 g, 62% from Boc-His(t-Ac)-OMe).
1H NMR (DMSO-d6) 1.34 (s, 9H), 2.82–3.12 (m, 2H), 3.72 (s, 3H),
4.20 (br s, 1H), 4.33 (s, 2H), 5.37 (br s, 2H), 6.75 (s, 1H), 6.88
(d, J = 8.70 Hz, 2H), 7.15–7.25 (m, 3H), 7.73 (s, 1H).; 13C NMR
(DMSO-d6) 25.3, 28.2, 52.8, 55.1, 69.0, 73.2, 78.1, 113.7, 127.6,
127.9, 129.0, 129.5, 138.3, 155.4, 158.9, 173.3; ESI MS Calcd for
[C20H27N3O6 + H]+ 406.198, found 406.1.
Examination of His Racemization during Synthesis of the
Model Peptide, Z-Ala-His-Pro-OH
Starting with H-Pro-Trt(2-Cl) resin (0.29 g, 0.69 mmol/g), manual
peptide chain assembly was carried out using the protocol of
30-min coupling with Fmoc-amino acid/HCTU/6-Cl-HOBt/DIEA
(4/4/4/8 equiv with respect to the peptide resin, 0.2 M, 0–5 min
preactivation) in DMF. MW heating was performed in a 25-ml
polypropylene open vessel placed into the MW cavity of a
300 W single-mode manual MW peptide synthesizer (CEM,
Discover SPS) set at 50 or 80 ꢁC; power pulsing sequences of
30 W were used for His coupling steps (5 min). Fmoc deprotection
was carried out with 20% piperidine in DMF, followed by washing
with DMF (5 ꢀ 2 min). The final release of peptides was achieved
with TFA/triisopropylsilane/H2O (95/5/5) containing MeONH2.HCl
(5 equiv with respect to the peptide resin) at room temperature
for 1 h. The crude peptides were directly analyzed by HPLC and
ESI MS analyses.
Fmoc-His(p-MBom)-OH (1a)
To a vigorously stirred solution of Boc-His(p-MBom)-OH (10.0 g,
23.8 mmol) and 2,6-lutidine (33.3 ml, 191 mmol) in CH2Cl2
(75 ml) at 0 ꢁC was added TMSOTf (34.6 ml, 191 mmol). After
10 min, the ice bath was removed, and the mixture was stirred
at room temperature for 18 h. The reaction mixture was placed
again in an ice bath, and ice-cold water (50 ml) was added.
Subsequently, the mixture was washed with CHCl3 (2 ꢀ 10 ml).
The aqueous layer was added to DMF (50 ml) and treated with
Fmoc-OSu (8.03 g, 23.8 mmol) and stirred at room temperature
for 4 h. The solution was adjusted with Na2CO3 aq. to pH 7–8.
After removal of the solvent, the residue was washed with H2O.
The residue was triturated with CHCl3/MeOH and Et2O to give
Fmoc-His(p-MBom)-OH (11.8 g, 84% yield): 1H NMR (DMSO-d6)
2.90–3.17 (m, 2H), 3.71 (s, 3H), 4.15–4.38 (m, 6H), 5.38 (q, 2H,
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,
Loading of the Fmoc-His-OH Derivatives onto Wang Resin
Wang resin (4-methoxybenzylalcohol resin) (185mg, 1.00 mmol)
was left to swell in THF/DMF (3:1, 10 ml) for 1 h before adding the
Fmoc-His-OH derivative [Fmoc-His(p-MBom)-OH (1a) or Fmoc-His
(t-Trt)-OH (1.50 mmol)] and DCC (1.50mmol). The suspensions were
cooled to ꢂ15 ꢁC, and then, 0.06 eq of DMAP was added. The
reaction mixture was kept at ꢂ15 ꢁC with slight shaking for 4 h
and left in an ice bath overnight. Fmoc deprotection was carried
out with 20% piperidine in DMF, followed by washing with DMF
(5ꢀ 2 min). The resin was then filtered off and washed with DMF
and DCM. After an aliquot (ca. 50 mg) of the resin had been treated
with 6 ml of TFA/water (v/v, 95:5) for 1 h, the filtrate was evaporated
to give the residue, which was checked for optical purity by using
Merfey’s method [9].
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SYNTHESIS AND APPLICATION OF FMOC-HIS(p-MBOM)-OH
O
O
BocHN CHC OMe
CH2
O
BocHN CHC OH
CH2
R
BocHN CHC OMe
CH2
O
Cl
Ac2O
1)
2) NaOH aq.
N
N
O
N
NH
N
N
Ac
R
2
3
4
O
O
H2N CHC OH
FmocHN CHC OH
CH2
CH2
Fmoc-OSu
TMSOTf
2,6-lutidine
O
N
N
O
N
N
R
R
5
1
a: R = 4-MeO-
b: R = 2,4-(MeO)2-
c: R = 3,4-(MeO)2-
Scheme 1. Synthetic route of Fmoc-His(MBom)-OH (1a) and its related compounds (1b, 1c).
Synthesis of Cys-Ang II, Trp-Ang II, and NPW30 Related Peptides
O
FmocHN CH C OH
CH2
The protected Cys-Ang II, Trp-Ang II, NPW30, and its related
peptides were assembled with an ABI 433A (Forester, CA, USA)
using Fmoc strategy on Wang-PEG resin (0.37g, 0.27 mmol/g).
The peptide chain was elongated using the FastMocW protocol
of coupling with Fmoc-amino acid/HCTU/6-Cl-HOBt/DIEA (4/4/4/
8 equiv) in 1-methyl-2-pyrrolidinone. The following side-chain-
protected amino acids were used: Trp(Boc), Tyr(tBu), Asp(OtBu),
Lys(Boc), His(t-Trt)/His(p-MBom), Ser(tBu), Arg(Pbf), Cys(Trt), and
Thr(tBu). Final cleavage and deprotection of peptides were
achieved with TFA/triisopropylsilane/H2O (95/5/5) in the presence
of MeONH2.HCl (5equiv) at room temperature for 1 h. The crude
peptides were directly analyzed by HPLC and ESI MS analysis.
O
N
N
MeO
4-Methoxybenzyloxymethyl
(MBom)
1a
Figure 1. Structure of Fmoc-His(MBom)-OH (1a).
NPW30 (10–15): H-Tyr-His-Thr-Val-Gly-Arg-OH
selective removal of the Na-Boc group avoiding the use of acidic
conditions was achieved by treatment with TMSOTf/2,6-lutidine
to give 5a–c in quantitative yield [11], with the a-amino functions
being protected by the Fmoc group. It is known that transformation
of an N-Boc group to a TMS carbamate using TMSOTf/2,6-lutidine is
compatible with the presence of other acid sensitive groups, that is,
tBu esters and ethers [12]. The optical purities of 1a–c synthesized
in this manner were >99.9% as determined by Marfey’s method
after removing the protecting groups [9].
Analytical HPLC condition: linear gradient of solvent B in solvent
A, 10% to 60% over 25 min, retention time = 16.1 min, LRMS (ESI)
m/z Calcd for C32H49N11O9 ([M + H]+) 732.4, found 732.4.
Results and Discussion
Preparation of the His Derivatives
Each Np-protecting group was introduced by regioselective
alkylation of Boc-His(t-Ac)-OMe using the chloride of the respective
mono-methoxy and di-methoxy substituted benzyloxymethanols.
Removal of the methyl ester by saponification with NaOH aq gave
intermediates in the synthesis of the corresponding Fmoc deriva-
tives (Scheme 1). The combination of the protecting groups on
these intermediates, that is, Na-Boc and respective Np-groups,
facilitated purification by crystallization to separate the undesired
t-isomer from the product without resorting to column chromatog-
raphy, thus making it possible to use the present synthetic
procedure for large-scale production (Figures 1 and 2). Further-
more, recrystallization of these intermediates could be effectively
performed to remove the respective D-isomers arising from sapon-
ification [10]. To convert 4a–c into the corresponding Fmoc deriva-
tives 1a–c, the Na-Boc group on these intermediates had to be
selectively cleaved without affecting the Np-protecting groups,
which are acid-sensitive in the same way as the Boc group. Thus,
The stability and removability of these Np-protecting groups
under the standard conditions used for Fmoc-SPPS were exam-
ined by RP-HPLC and summarized in Table 1 in comparison with
those of the Trt group. The MBom, 2,4-DMBom, and 3,4-DMBom
groups were found to be completely stable during the repetitive
Fmoc deprotection reaction using 20% piperidine in DMF but
readily removable by TFA in the same manner as the Trt group.
Among them, the MBom group was the only protecting group
that remained attached to the imidazole ring during detachment
of the protected peptide segments with a free carboxyl group
from the Trt(2-Cl) resin by treatment with 1% TFA/DCM or
DCM/trifluoroethanol/AcOH (v/v, 3/1/1) [13]: The Trt group is
known to be susceptible to this condition. When applying
the protected His-containing peptide segments prepared on
acid-sensitive linkers for the convergent synthesis, partial loss of
the Nim-protecting group on the His residue may not only
J. Pept. Sci. 2012; 18: 763–769 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

HIBINO, MIKI AND NISHIUCHI
Figure 2. HPLC profiles of the products, Boc-His(p-MBom)-OH (4a). (A) Crude product without purification; (B) product obtained after crystallization from CHCl3/
AcOEt; and (C) mother solution. Eluent: 10–60% MeCN in 0.1% TFA. Peaks 1 and 2 indicate Boc-His(t-MBom)-OH and Boc-His(p,t-diMBom)-OH, respectively.
hamper their purification procedure but also cause side reactions
involving the nucleophilicity of the imidazole ring in the subse-
quent segment condensation. These results clearly indicated that
as a carboxyl component, this protocol is known to be more
or less accompanied by their racemization in correlation with
the length of preactivation [14]. The rate of racemization of
Fmoc-His(t-Trt)-OH during the activating procedure significantly
increased in a time-dependent fashion because of its unpro-
tected p-nitrogen, which is involved in promoting racemization
pathways, whereas that of Fmoc-His(p-MBom)-OH increased very
slightly, and it was comparable with those observed with other
ordinary Fmoc-amino acids (Table 2) [15]. As long as the duration
of preactivation was limited to no more than 1 min, which is the
standard condition, Fmoc-His(p-MBom)-OH was found to be
accompanied by virtually no racemization on activating and
coupling steps of the conventional SPPS compared with that of
Fmoc-His(t-Trt)-OH (2.9%). This indicates that the Np-MBom
group would effectively prevent racemization even in the case
of a slow coupling process where the amino component is
sterically hindered [16]. Furthermore, Fmoc-His(p-MBom)-OH
the MBom group for the His residue can offer permanent Nim
-
protection throughout the synthesis of the protected peptides.
Application of Fmoc-His(p-MBom)-OH in SPPS
Suppressive effect of Np-MBom on racemization
The suppressive effect of the Np-MBom group on racemization
during the incorporation of His was evaluated by synthesizing a
model peptide, Z-Ala-His-Pro-OH [10], via the conventional and
the MW-assisted SPPS. The peptide chain was elongated onto
an H-Pro-Trt(2-Cl) resin by the widely accepted protocol of
coupling with Fmoc (or Z)-amino acid/HCTU/6-Cl-HOBt/DIEA
(4/4/4/8 equiv) in DMF. Upon activation of amino acid derivatives
Table 1. Stability of Fmoc-His(X)-OH under detachment conditions using a weak acid
Imidazole protection
2,4-DMBom 3,4-DMBom
Conditions
X =
p-MBom
t-Trt
1% TFA/CH2Cl2
rt 3 h
rt 3 h
100
100
87
94
92
72
88
DCM/trifluoroethanol/AcOH(v/v, 3/1/1)
100
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SYNTHESIS AND APPLICATION OF FMOC-HIS(p-MBOM)-OH
Table 2. Racemization of His during the synthesis of the model pep-
tide, Z-Ala-His-Pro-OH, as a function of the length of preactivation
with HCTU
Preactivation (min)
X =
Racemization (%)a
p-MBom
t-Trt
0
1
5
<0.1b
<0.1b
0.3
1.0
2.9
7.8
aDefined as (Z-Ala-D-His-Pro)/(Z-Ala-L-His-Pro) ꢀ 100.
bBelow detection limit.
provided significant reduction in the level of racemization even
when performing the MW-assisted SPPS at 50 and 80 ꢁC (0.2%
and 0.5%, respectively), whereas Fmoc-His(t-Trt)-OH led to a
considerable extent (3.7% and 16.6%, respectively) as shown in
Table 3. MW-assisted SPPS is accepted as a valid support of
enhancement of the coupling efficiency in a short time. The
coupling reactions are usually carried out under MW irradiation
at a high temperature of around 80 ꢁC [17]. However, recent
publications have drawn attention to the susceptibility to racemi-
zation of Fmoc-His(t-Trt)-OH under these conditions [5,18].
Thus, it is recommended that incorporation of Fmoc-His(t-Trt)-
OH onto a peptide chain be carried out at 50 ꢁC or less, although
there is no advantage in terms of coupling efficiency over
the procedure performed at 80 ꢁC. In contrast, Fmoc-His
(p-MBom)-OH proved to reduce the rate of racemization to an
acceptable level even when performing MW-assisted SPPS at
80 ꢁC (Figure 3). This measure using Fmoc-His(p-MBom)-OH can
facilitate the exclusion of racemization with His during the course
of chain assembly by MW-assisted SPPS.
The efficient acylation of hydroxyl-functionalized solid supports
such as Wang resin with Fmoc-protected amino acids without race-
mization is the essential first step for SPPS. Esterification of the His
derivatives with the aid of the DCC/DMAP method to a hydroxyl
group on the resin is known to be accompanied by severe racemi-
zation [19], although several methods involving activation of the
hydroxyl group for linking to Fmoc amino acids, for example, the
use of trichloroacetimidate Wang resin and the Mitsunobu reaction,
are available to avoid the side reaction with racemization [20,21].
Thus, we tried to apply the DCC/DMAP method for loading Fmoc-
His(p-MBom)-OH to Wang resin in order to evaluate the optical sta-
bility during this process. Fmoc-His(p-MBom)-OH caused a dramatic
reduction in the level of racemization during its loading, whereas
Fmoc-His(t-Trt)-OH led to a serious level of racemization (Table 4).
Figure 3. HPLC profiles of the products, Z-Ala-His-Pro-OH, obtained by
performing MW-assisted SPPS at 80 ꢁC. Incorporation of His was performed
by coupling Fmoc-His(t-Trt)-OH (A) and Fmoc-His(p-MBom)-OH (B). Eluent:
15–40% MeCN in 0.1% TFA.
species, a methoxybenzyl cation, from the MBom group occurs in
side reactions. Formaldehyde can lead to hydroxymethylated
modification of a-amino and e-amino groups, although the extent
is inconsequential. On the other hand, when a Cys or a Trp residue
is located at the N-terminus, formaldehyde can react with it to
produce a Thz-peptide or THCar-peptide, respectively, during isola-
tion from an acidolytic mixture [22,23]. The methoxybenzyl cation
can cause alkylation of susceptible residues such as Cys and Trp
[24]. Thus, we applied Fmoc-His(p-MBom)-OH to the synthesis of
Cys-Ang II (CDRVYIHPF) and Trp-Ang II to examine their by-product
formation associated with the use of this protecting group as
summarized in Tables 5 and 6, respectively. As has been reported,
the formation of Thz-peptide or THCar-peptide arising from the
generation of formaldehyde during TFA treatment was almost
completely suppressed by the addition of methoxyamine hydro-
chloride (MeONH2ꢃHCl, 5 equiv) to the reaction mixture [10,25].
The alkylation of Trp with a carbocation evolved from the MBom
group was negligible in amount as long as its indole ring was
protected by the Boc group, which is known to produce the
Table 4. Racemization during the loading of Fmoc-His(X)-OH onto
Wang resin
X
Racemization (%)
t-Trt
p-MBom
31.4
0.3
Final deprotection of His(p-MBom)-containing peptides
After the final TFA deprotection and cleavage of the peptide resin,
the generation of formaldehyde and an electrophilic-alkylating
Table 5. Effect of additives on Cys modification during TFA
deprotection
Table 3. Racemization of His during MW-assisted SPPS of the model
peptide, Z-Ala-His-Pro-OH, as a function of the coupling temperature
Ratio of Cys-Ang II to Cys(X)-Ang II
Cys-Ang II
Thz-Ang II
Cys(MeOBzl)-
Ang II
Temperature (ꢁC)
X =
Racemization (%)
Additives
p-MBom
t-Trt
None
88
91
99
5.3
<1.0
<1.0
7.1
7.8
50
80
0.2
0.5
3.7
MeONH2ꢃHCl
MeONH2ꢃHCl/PhSH
16.6
<1.0
J. Pept. Sci. 2012; 18: 763–769 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

HIBINO, MIKI AND NISHIUCHI
Table 6. Effect of additives on Trp modification during TFA
deprotection
differ from those of the natural product, extreme care is required in
their chemical synthesis, especially of biologically active peptides,
to avoid ambiguity in the quality of the synthesized peptides.
Therefore, it is important to exclude the risk of racemization
with incorporation of His by using Fmoc-His(p-MBom)-OH during
the course of the chain assembly. No racemization with His11 was
found in the segment (10–15) on synthesis using Fmoc-His
(p-MBom)-OH.
Ratio of Trp-Ang II to Trp(X)-Ang II
Additives
Trp-Ang II THCar-Ang II Trp(MeOBzl)-Ang II
None
90
98
99
7.8
<1.0
<1.0
1.2
1.2
MeONH2ꢃHCl
MeONH2ꢃHCl/PhSH
<1.0
Nin-carbamic acid to prevent such electrophilic additions during
TFA treatment (Table 6) [26]. In contrast, performing TFA cleavage
in the presence of thiol compounds was necessary to avoid the
alkylation of Cys (Table 5). This measure using MeONH2ꢃHCl and
thiols could effectively prevent the side reactions associated with
the use of the MBom group.
Conclusions
The MBom group proved to be an ideal protecting group for the His
residue in terms of offering Np-protection and Fmoc compatibility to
eliminate side-chain-induced racemization during incorporation of
His. The suppressive effect of Np-MBom on racemization occurred
even in the case of MW-assisted SPPS performed at 50 and 80ꢁC.
In particular, the MBom group was shown to be a suitable protecting
group for the convergent synthesis because it remains attached to
the imidazole ring during detachment of the protected peptide
segments from acid-sensitive linkers. Furthermore, we were able to
confirm the applicability of the present synthetic procedure of
Fmoc-His(p-MBom)-OH for large-scale production.
Synthesis of rat neuropeptide W-30
To demonstrate the usefulness of the Np-MBom group on His, we
synthesized rat NPW30 (WYKHVASPRY10HTVGRASGLL20MGLRR-
SPYLW30), a food intake-regulating peptide [27], by Fmoc SPPS
using a 1-min preactivation procedure of coupling with Fmoc-amino
acid/HCTU/6-Cl-HOBt/DIEA (4/4/4/8equiv) in DMF/1-methyl-2-
pyrrolidinone. When synthesizing NPW30 by using Fmoc-His
(t-Trt)-OH, the product was found to be contaminated by 4% of
D-His4-diastereoisomer at a moderate estimate; this could be only
detected as a shoulder peak by RP-HPLC when an isocratic system
was used. The other isomer, D-His11-diastreoisomer, was eluted
at the same retention time as the desired peptide on RP-, ion
exchanged HPLC, and capillary zone electrophoresis, although
RP-HPLC analysis of the segment (10–15) obtained by digesting
the synthetic NPW30 with trypsin revealed that racemization had
occurred at His11 (3.2%) as shown in Figure 4. Thus, there were
no practical purification procedures available for removing the
racemized peptides arising from D-His4 or D-His11. Because it is
known that a diastereoisomer of biologically active peptides with
racemization somewhere in its sequence may pose the risk of
developing biological efficacy and/or pharmacological targets that
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J. Pept. Sci. 2012; 18: 763–769 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci