Preparation of a core-shell type MBHA resin and its application for solid-phase peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2009.05.009

MBHA (4-methylbenzhydrylamine) resin has been extensively used as a solid support for the synthesis of peptide amides. Herein, we prepared the core-shell-type MBHA resin by benzotriazole-catalyzed amidoalkylation and partial hydrolysis. The core-shell str

Full text of DOI:10.1016/j.tetlet.2009.05.009

Tetrahedron Letters 50 (2009) 4272–4275
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Preparation of a core–shell type MBHA resin and its application
for solid-phase peptide synthesis
Jeong-Hyun Choi a, Tae-Kyung Lee a, Jang-Woong Byun b, Yoon-Sik Lee a,
*
a School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea
b BeadTech Inc., Institute for Chemical Processes, Seoul National University, Seoul 151-744, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
MBHA (4-methylbenzhydrylamine) resin has been extensively used as a solid support for the synthesis of
peptide amides. Herein, we prepared the core–shell-type MBHA resin by benzotriazole-catalyzed amido-
alkylation and partial hydrolysis. The core–shell structure of the MBHA resin was confirmed by two-pho-
ton microscopy and its synthetic performance in solid-phase peptide synthesis (SPPS) was evaluated.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 6 March 2009
Revised 30 April 2009
Accepted 7 May 2009
Available online 10 May 2009
Keywords:
MBHA resin
Core–shell-type resin
a-Amidoalkylation
Partial hydrolysis
Solid-phase peptide synthesis
Recently various polymeric supports have been widely used in
solid-phase peptide synthesis (SPPS) and their characteristics are
important for improving synthetic efficiency. This is especially true
for the distribution of the functional groups within the resin, since
this characteristic greatly affects the diffusion of the reagents.
Functional groups in the surface layer of the resin are more acces-
sible to the reactants than those in the core layer. Therefore, so-
called ‘core–shell’ type resins, in which most of the reactive sites
are located in the surface layer, were developed for better solid-
phase reactions. Several types of core–shell type resins have re-
cently been developed and used in SPPS, such as CutiCore resin,1
HiCore resin,2 AM SURETM resin,3,4 and 2-chlorotrityl chloride
(CTC) resin,5 and these resins displayed better synthetic efficiency
than conventional ones.
MBHA (4-methylbenzhydrylamine) resin is a well-known solid
support for the synthesis of peptide amides and its industrial de-
mand for peptide production is currently increasing. In order to
prepare MBHA resin, many methods have been developed for
introducing the benzhydrylamine (BHA) structure into the polysty-
rene resin, such as the oxime group reduction method,6 Leuckart
reduction method,7 phthalimido linker method,8 and imine group
reduction method.9,10 However, these methods have some draw-
backs including low conversion and harsh reaction conditions.
In a previous study, we presented a simple method for the syn-
thesis of the MBHA resin.11 Compared to these other methods,
MBHA resin was easily prepared by benzotriazole-mediated amido-
alkylation. First, formamido PS resin, the precursor of MBHA resin,
was synthesized from 1% DVB-PS resin using bis(formamide) and
1-benzotriazole. MBHA resin was efficiently obtained by removing
the formamido groups and it displayed excellent properties as a so-
lid support. In this Letter, we present the preparation of the core–
shell-type MBHA resin and examine its novel characteristics, which
result from the distribution of functional groups. Furthermore, we
report the synthetic efficiency of the core–shell-type MBHA resin
in SPPS compared to that of the non-core–shell type.
Formamido PS resin (1) was synthesized using our method for
the preparation of MBHA resin, and the core–shell structure of
the MBHA resin (2) was constructed by partial hydrolysis of the
formamido groups in an aqueous solvent (Scheme 1). Since the
core layer of the hydrophobic PS resin was not readily accessible
to the hydrophilic reagents during hydrolysis, the formamido
groups (1) were partially hydrolyzed in 6 N HCl/EtOH at 50 °C for
3 h, and converted into amino groups only on the surface layer of
the resin. The loading capacity of the core–shell type MBHA resin
(2) was determined to be 0.58 mmol/g by Fmoc-titration after
loading Fmoc-Gly-OH onto the resin, and the existence of both
amine and formamide groups was confirmed by FT-IR analysis.
To assess the distribution of the functional groups within the
resin, we coupled a fluorescent dye (dansyl chloride) to the amino
groups of the core–shell type MBHA resin, and imaged it by two-
photon microscopy (TPM, emission 777 nm).12–15 As shown in
the TPM images of the core–shell type MBHA resin (Fig. 1), fluores-
cence was detected only at the shell layer and not in the core
* Corresponding author. Tel.: +82 2 880 7080; fax: +82 2 876 9625.
E-mail address: yslee@snu.ac.kr (Y.-S. Lee).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.05.009

J.-H. Choi et al. / Tetrahedron Letters 50 (2009) 4272–4275
4273
H
O
H
H
N
H
N
O
H
a
b
NH2
N
O
+
H
H
N
O
H
1% DVB-PS
Bis(formamide)
1
2
Scheme 1. Preparation of the formamido PS resin (1) and core–shell type MBHA resin (2). Reagents and conditions: (a) 1-benzotriazole (0.2 equiv based on the
bis(formamide) linker), AlCl3, DCE, reflux, 72 h; (b) 6 N HCl, EtOH, 50 °C, 3 h.
Figure 1. Distribution of functional groups in core–shell type MBHA resin: Two-photon microscopic fluorescence images of dansyl chloride-coupled resin; Images on the
lefthand side are taken from a single bead. The graph on right-hand side is the corresponding intensity values for a horizontal line passing through the center of the resin.
region, indicating that the resin (2) contained the core–shell struc-
ture. In contrast, fluorescence was observed in all regions of the
non-core–shell type MBHA resin. This indicates that functional
groups were distributed within the core layer of the non-core–shell
type resin, which may cause kinetic problems when bulky reagents
are added during the reaction (see Supplementary data).
To determine the loading efficiency of Fmoc-amino acids
(Fmoc-AAs) onto the core–shell type MBHA resin (2), 20 different
Fmoc-AAs were introduced onto resin (2) using HBTU and HOBt
for 2 h, and the resulting resins were checked by Fmoc-titration.
Their loading levels were 0.45–0.58 mmol/g and the loading yields
were 78% to quantitative (Table 1). These results indicate that resin
(2) had a high loading capacity and excellent performance in the
first loading step.
In SPPS, this first amino acid loading step is rather slow due to
the matrix effect of the polymer backbone. However, if the core–
shell type resin can minimize this problem, the coupling reaction
could be performed much more efficiently. Thus, we examined
whether the coupling rate of the first amino acid to the core–shell
type resin was faster than that of other resins. In order to deter-
mine this, Fmoc-His(Trt)-OH and Fmoc-Phe-OH were selected be-
cause Fmoc-His(Trt)-OH had the lowest loading yield (78%) and
We investigated the chemical stability of the formamido groups
that remained in the core layer of the core–shell type MBHA resin.
The formamido PS resin (1) was subjected to several strong acidic
and basic conditions including a typical peptide synthesis condi-
tion, and any changes in the functional groups were determined
by FT-IR analysis. The intensity of the carbonyl band (1684 cmÀ1
)
was maintained after all treatment conditions, and there were no
significant loss or change in the formamido groups. Therefore, we
concluded that the formamido groups do not cause any side reac-
tions during peptide synthesis.
Table 1
Loading levels and yields of Fmoc-amino acids onto the core–shell type MBHA resin (2): Fmoc-amino acids were loaded onto the resin using HBTU, HOBt, and DIPEA in NMP for
2 h at 25 °C
Fmoc-AA-OH
Loading level (mmol/g)
Loading yield (%)
Fmoc-AA-OH
Loading level (mmol/g)
Loading yield (%)
Ala
0.53
0.45
0.46
0.49
0.48
0.47
0.51
0.53
0.45
0.51
91
78
79
84
82
81
88
91
78
88
Leu
Lys(Boc)
Met
Phe
Pro
Ser(tBu)
Thr(tBu)
Trp(Boc)
Tyr(tBu)
Val
0.57
0.55
0.57
0.58
0.55
0.57
0.55
0.56
0.54
0.56
98
94
98
Arg(Pbf)
Asn(Trt)
Asp(tBu)
Cys(Trt)
Gln(Trt)
Glu(tBu)
Gly
Quantitative
95
99
94
96
93
96
His(Trt)
Ile

4274
J.-H. Choi et al. / Tetrahedron Letters 50 (2009) 4272–4275
Figure 2. Coupling kinetics of Fmoc-amino acid loading onto the resins: The coupling was proceeded with HBTU, HOBt, and DIPEA in NMP during 5 min, 15 min, 30 min,
60 min, and 90 min. (a) Fmoc-His(Trt)-OH loading; (b) Fmoc-Phe-OH loading.
Table 2
Fmoc-Phe-OH showed the highest loading yield (quantitative).
Swelling volume of the MBHA resins in typical solvents
Three types of MBHA resins, core–shell type (0.58 mmol/g), non-
core–shell type (0.74 mmol/g), and a commercially available one
Swelling volume (mL/g resin)
(1.20 mmol/g), were used in the coupling kinetics study. In order
to set similar capacities of resins, Fmoc-Rink amide linker was an-
chored onto each of the MBHA resins on different conditions,
resulting in Fmoc-Rink-amide-MBHA-cs-resin (0.40 mmol/g) and
Fmoc-Rink-amide MBHA resin (0.43 and 0.50 mmol/g). After
adjusting almost similar capacities, Fmoc-amino acids were loaded
onto the resins to check the first amino acid coupling rate. The cou-
pling yields were examined by Fmoc-titration. As expected, the
coupling rate of the core–shell type resin was faster than that on
the non-core–shell resin (Fig. 2). These results clearly demonstrate
that the first amino acid coupling reaction was faster and more
efficient in the core–shell type MBHA resin (2) than the non-
core–shell type resin (see Supplementary data).
We also found that the core–shell type MBHA resin (2) was less
swollen in typical solvents such as DMF, NMP, DCM, THF, and
MeOH than in the non-core–shell type MBHA resin (Table 2). In
general, the high swelling properties of the resin in organic sol-
vents are important factors because it is closely related to the dif-
fusion of reagents within the resin and hence affects the reaction
efficiency in SPPS. However, during the manufacturing processes
for peptide synthesis, this causes an excessive consumption of sol-
vents and problems associated with waste disposal. Therefore, the
lower swelling properties of the core–shell type MBHA resin are
advantageous in that they will consume less solvent during pep-
tide production. In addition, the core–shell type MBHA resin dis-
DMF
NMP
CH2Cl2
THF
CH3OH
Core–shell type MBHA resin
Non-core–shell type MBHA resin
5.6
6.0
6.4
7.2
4.4
6.4
5.2
6.8
2.7
2.8
played better synthetic performance due to the fast kinetics of
reagent diffusion.3,4
To test the synthetic performance of the core–shell type MBHA
resin (2), the human myelin basic protein fragment (MBP (87–99)),
which has been industrially synthesized from a MBHA resin, was
selected as a model protein fragment. A Fmoc-Rink amide linker
was anchored onto the MBHA resins such that the Fmoc/tBu strat-
egy could be used for peptide synthesis. After MBP (87–99) was
synthesized on the Fmoc-Pro-NH-Rink amide-MBHA-cs-resin
(0.40 mmol/g resin), the peptide fragments were cleaved with
TFA/TIS/H2O (95:2.5:2.5), and the purity was determined by HPLC
(Fig. 3). The product showed one major peak, which was confirmed
to be MBP (87–99) by MALDI-TOF Mass. The desired peptide was
obtained in 83% purity from the core–shell type MBHA resin (2),
which was slightly higher than that obtained from the non-core–
shell type resin (79%).
In summary, the core–shell type MBHA resin (2) was efficiently
prepared by partial hydrolysis of the formamido groups on the sur-
face layer, and resin (2) displayed good physical and chemical
properties as a solid support in SPPS. It also had a better synthetic
Figure 3. HPLC analyses of the crude products MBP (87–99) synthesized from (a) core–shell type MBHA resin; (b) non-core–shell type MBHA resin (MALDI-TOF: calcd 1554.9
for VHFFKNITPRTP-NH2 [M+H]+, found 1555.0).

J.-H. Choi et al. / Tetrahedron Letters 50 (2009) 4272–4275
4275
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Acknowledgments
This study was supported by a grant from the Korea Health 21
R&D Project, Ministry of Health & Welfare, Republic of Korea
(A050432), and Seoul R&D Program (10538), through Institute of
Bioengineering, Seoul National University, Seoul, Korea.
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Supplementary data
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Supplementary data associated with this article can be found, in
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