A novel protecting group for constructing combinatorial peptide libraries

Article abstract of DOI:10.1246/bcsj.74.733

3,4,5-Tris(octadecyloxy)benzyl alcohol, HO-Bzl(OC₁₈)₃, was prepared from gallic acid and stearyl bromide. Using conventional step-wise elongation, N,C-protected peptides, Fmoc - AA_n - ... - AA₁ - OBzl(OC₁₈)₃, were synthesized. The substituted benzyl esters were selectively cleaved by a treatment with 4 M hydrogen chloride in ethyl acetate to give Fmoc - AA_n - ... - AA₁ - OH and HO - Bzl(OC₁₈)₃. Thus, the substituted benzyl group is effective for the protection of C-terminal carboxyl groups in liquid-phase peptide synthesis. Because the substituted benzyl group has a moderately high molecular weight, Fmoc - AA_n - ... - AA₁ - OBzl(OC₁₈)₃ can be easily purified by size-exclusion chromatography; all protected peptides are eluted in the void fraction of a Sephadex LH-20 gel-filtration column. The combination of the carboxyl-protecting group Bzl(OC₁₈)₃ with simple purification by the gel-filtration gives a novel route for constructing combinatorial peptide libraries in the solution phase.

Full text of DOI:10.1246/bcsj.74.733

c. Jpn.
© 2001 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 74, 733—738 (2001)
733
e Chemical
ciety of Ja-
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e Chemical
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n
A Novel Protecting Group for Constructing Combinatorial Peptide
Libraries
Novel Protecting Group for Peptide Library
H. Tamiaki et al.
Hitoshi Tamiaki,* Tomoyuki Obata, Yasuo Azefu, and Kazunori Toma†
01
3
8
Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University,
Kusatsu, Shiga 525-8577
SJA8
09-2673
317
†The Noguchi Institute, Itabashi, Tokyo 173-0003
(Received September 13, 2000)
ptember
00
3,4,5-Tris(octadecyloxy)benzyl alcohol, HO–Bzl(OC18)3, was prepared from gallic acid and stearyl bromide. Us-
ing conventional step-wise elongation, N,C-protected peptides, Fmoc–AAn–...–AA1–OBzl(OC18)3, were synthesized.
The substituted benzyl esters were selectively cleaved by a treatment with 4 M hydrogen chloride in ethyl acetate to give
Fmoc–AAn–...–AA1–OH and HO–Bzl(OC18)3. Thus, the substituted benzyl group is effective for the protection of C-ter-
minal carboxyl groups in liquid-phase peptide synthesis. Because the substituted benzyl group has a moderately high
molecular weight, Fmoc–AAn–...–AA1–OBzl(OC18)3 can be easily purified by size-exclusion chromatography; all pro-
tected peptides are eluted in the void fraction of a Sephadex LH-20 gel-filtration column. The combination of the car-
boxyl-protecting group Bzl(OC18)3 with simple purification by the gel-filtration gives a novel route for constructing com-
binatorial peptide libraries in the solution phase.
00
.4
Combinatorial chemistry has attracted much attention in
various fields from lead compound searches in drug discovery1
to host–guest chemistry as a means of chemical evolution.2
Peptide libraries are the most classic example of combinatorial
libraries, and synthetic peptide libraries are usually prepared in
the solid phase.3 Peptide synthesis on a polymer support is
well established and the procedures are simple, including easy
separation of the desired peptides from the reaction mixture by
washing with a solvent. However, solid-phase synthesis has
unavoidable problems: the difficulties in reacting the soluble
reagents with all the reactive sites on the solid support and in
monitoring the heterogeneous reaction. To overcome these
problems, soluble supports have been proposed, such as a
polyethylene glycol4 and a dendrimer.5 As polyethylene glycol
is a mixture of homologs with a range of molecular weights,
this heterogeneity makes monitoring reactions difficult: typi-
cally, chromatographic separation as well as mass spectral
analysis is complicated. A dendrimer has a uniform molecular
structure, but has multiple reactive sites in the molecule, which
make completing as well as monitoring reactions somewhat
difficult.
Here, we report on the preparation of a novel protecting
group, 3,4,5-tris(octadecyloxy)benzyl alcohol 2, for peptide
synthesis in the solution phase. The substituted benzyl alcohol
2 is effective for protecting the carboxyl group in a convention-
al peptide synthesis using the Fmoc strategy. The N,C-protect-
ed peptides have well-defined molecular structures and the re-
action is easily monitored by standard techniques used in
organic synthesis, including chromatography, mass and NMR
spectroscopies. Moreover, the protected peptides are fairly
large molecules (MW > 1000) and are able to be easily purified
by gel filtration. The desired peptides are eluted in the void
fraction using size-exclusion chromatography. The combina-
tion of 2 as a carboxyl protecting group with simple purifica-
tion by gel filtration gives a novel route for constructing syn-
thetic peptide libraries.6
Results and Discussion
Several protecting groups for the carboxyl group in liquid-
phase peptide synthesis are available:7 the acid-labile t-butyl
group and the benzyl group cleavable by hydrogenation are
generally used. Substituted benzyl esters have also been inves-
tigated as protecting groups. The stability is dependent upon
the substituents:7 electron-withdrawing groups on the benzene
ring stabilize the esters to acidic conditions and electron-do-
nating groups make the esters more acid-labile. To prepare an
acid-labile carboxyl protecting group with a fairly high molec-
ular weight, a 3,4,5-trialkoxybenzyl alcohol was investigated.
The starting material, gallic acid, is inexpensive and has many
reactive sites on the benzene ring. Saturated straight alkyl
chains, –(CH2)nꢀ1CH3, are chemically stable and the long alkyl
chains increase the molecular weight. Octadecyl groups (n ꢁ
18) were used in the present study. Inexpensive and commer-
cially available stearyl bromide is effective as an octadecyl-in-
troducing reagent to all three phenolic hydroxy groups of gal-
lic acid.
3,4,5-Tris(octadecyloxy)benzyl alcohol (HO–Bzl(OC18)3, 2)
was prepared from gallic acid and stearyl bromide as shown in
Scheme 1. Percec and his colleagues have already reported the
preparation of 3,4,5-tris(dodecyloxy)benzyl alcohol,8 and we
slightly modified their procedures (see Experimental section).
The synthetic procedures are so simple that the large-scale
preparation of 2 is possible without any difficulty.
Because 3,4,5-tris(octadecyloxy)benzyl esters have elec-

734 Bull. Chem. Soc. Jpn., 74, No. 4 (2001)
Novel Protecting Group for Peptide Library
Scheme 1.
Synthesis of carboxyl-protecting substituted benzyl alcohols.
tron-donating groups, they are expected to be hydrolyzed by a
treatment with acid, as described above. Therefore, the base-
labile and acid-resistant 9-fluorenylmethyloxycarbonyl (Fmoc)
group was used for protection of the amino groups in the pep-
tide syntheses. Fmoc-Ala-OH reacted with HO–Bzl(OC18)3
(2) in dichloromethane with 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide (EDC) as the coupling reagent in the presence
of 1-hydroxybenzotriazole (HOBt) to give Fmoc–Ala–O–
Bzl(OC18)3 in 63% isolated yield (Scheme 2). The Fmoc pro-
tecting group was selectively cleaved by the action of piperi-
dine in dichloromethane at 0 ˚C to give H–Ala–OBzl(OC18)3 in
92% isolated yield. The reaction of Fmoc–Phe–OH with H–
Ala–OBzl(OC18)3 by EDC–HOBt afforded Fmoc–Phe–Ala–
OBzl(OC18)3 in 67% isolated yield. Fmoc-deprotection of the
dipeptide and successive peptide formation with Fmoc–Leu–
OH similarly yielded the protected tripeptide Fmoc–Leu–Phe–
Ala–OBzl(OC18)3. All of the synthetic compounds were puri-
fied by silica-gel chromatography and/or recrystallization and
characterized by their H NMR, infra-red and/or mass spec-
1
tra. All of the compounds were pure enantiomers or diastere-
omers, indicating that no racemization occurred. It is to be
noted that all of the reactions were clean, and the yields esti-
1
mated from TLC and H NMR analyses were higher (> 90%)
than the isolated yields described above.
Cleavage of the ester bond of the 3,4,5-tris(octadecyl-
oxy)benzyl esters was examined under several conditions (see
Scheme 3). The treatment of Fmoc–Ala–OBzl(OC18)3 by hy-
drogen bromide in acetic acid induced a selective cleavage of
the ester to give Fmoc–Ala–OH without any loss of the Fmoc
group. Rather than hydrolyzed HO–Bzl(OC18)3, the corre-
sponding bromide Br–Bzl(OC18)3 was isolated. When Fmoc–
Ala–OBzl(OC18)3 was subjected to less acidic conditions, such
Scheme 2.
Synthesis of peptides Fmoc–AAn–...–AA1–OBzl(OC18)3.

H. Tamiaki et al.
Bull. Chem. Soc. Jpn., 74, No. 4 (2001) 735
Scheme 3.
Cleavage of protecting groups in Fmoc–AAn–...–AA1–OBzl(OC18)3.
gy for constructing combinatorial peptide libraries.
as 4 M hydrogen chloride in ethyl acetate (1 M ꢁ 1 mol
dmꢀ3),9 the quantitative hydrolysis afforded a 1:1 mixture of
Fmoc–Ala–OH and HO–Bzl(OC18)3. After evaporation, the
resulting residue was treated with hexane and insoluble Fmoc–
Ala–OH was isolated as a pure white solid by filtration. The
filtrate was evaporated to give pure HO–Bzl(OC18)3, which
could be re-used for protection as it stood. The acidic hydroly-
sis procedure is very simple and this acidic cleavage was ap-
plied to other peptides. Fmoc–Phe–Ala–OBzl(OC18)3 and
Fmoc–Leu–Phe–Ala–OBzl(OC18)3 were hydrolyzed by stir-
ring in 4 M HCl–AcOEt overnight and successively evaporated
in vacuo to give Fmoc–Phe–Ala–OH and Fmoc–Leu–Phe–
Ala–OH, respectively, plus HO–Bzl(OC18)3. Hydrogenation of
Fmoc–Ala–OBzl(OC18)3 on palladium–charcoal in tetrahydro-
furan induced deprotection of both the Fmoc and Bzl(OC18)3
groups to give unprotected alanine in one step. After hydroge-
nation, 3,4,5-tris(octadecyloxy)toluene, H–Bzl(OC18)3, was
isolated, and thus could not be re-used as a protecting reagent.
The Fmoc–Ala–OH and H–Ala–OH formed were both in their
L-forms and no racemization was observed during the above-
mentioned deprotections. Thus, the 3,4,5-tris(octadecyl-
oxy)benzyl group has been proven to be an effective carboxyl
protecting group in peptide synthesis.
In the peptide synthesis described above, the resulting N,C-
protected peptides (or amino acid) Fmoc–AAn–…–AA1–O–
Bzl(OC18)3 were purified by silica-gel chromatography. These
peptides have fairly large molecular weights because of the C-
protected Bzl(OC18)3 group. We therefore examined the idea
of purifying the peptides by size-exclusion chromatography
with Sephadex LH-20 from Amersham Pharmacia Biotech, a
hydroxypropylated cross-linked dextran gel in organic sol-
vents. After complete consumption of an amino component
(checked by TLC), the dichloromethane solution containing
the reaction mixture was loaded onto a LH-20 gel column.
The column was eluted with dichloromethane and the first void
fraction was collected and evaporated to give a white solid.
The 1H NMR and mass spectra showed that the solid was sole-
ly the desired peptides, indicating that such a gel filtration was
an extremely simple purification for all the Fmoc–AAn–…–
AA1–OBzl(OC18)3 peptides prepared.
At 914, the molecular weight of HO–Bzl(OC18)3 (2) is not
very high. A larger benzyl alcohol, 3,4,5-tris[3,4,5-tris(octade-
cyloxy)benzyloxy]benzyl alcohol (HO–Bzl[OBzl(OC18)3]3, 4,
MW 2871), was prepared from the coupling of methyl gallate
and Cl–Bzl(OC18)3 (3) with successive LiAlH4-reduction in
90% isolated yield for the two steps by a slight modification of
the reported procedures.8 Fmoc–Ala–OH reacted with HO–
Bzl[OBzl(OC18)3]3 (4) by EDC–HOBt to give Fmoc–Ala–OB-
zl[OBzl(OC18)3]3 in 30% isolated yield. The low yield is as-
cribed to the low reactivity of the sterically hindered alcohol.
The reactivity should increase by using spacers, as in commer-
cially available polymer supports.10
Because the benzyl group is effective for the protection of
hydroxy and mercapto groups,7 the Bzl(OC18)3 group should
also be useful for the protection of OH and SH as well as
COOH. Indeed, Cl–Bzl(OC18)3 reacted smoothly with methyl
gallate, as described above. Moreover, 3,4,5-tris(octadecy-
loxy)benzoic acid, prepared by the hydrolysis of methyl 3,4,5-
tris(octadecyloxy)benzoate (1) with KOH (89%), should be
useful for protection of hydroxy groups through the formation
of an ester bond and 3,4,5-tris(octadecyloxy)benzaldehyde,
prepared by the oxidation of HO–Bzl(OC18)3 (2) with pyridini-
um chlorochromate (87%), should be useful for the protection
of amino groups through the formation of an imino bond. All
of the protected compounds proposed above would be purified
by a simple gel filtration as well. Although we have demon-
strated only peptide syntheses, the Bzl(OC18)3 group together
with these derivatives described above should be applicable to
the construction of many small molecule libraries.
Experimental
General. All melting points were measured with a Yanagi-
moto micro melting apparatus and are uncorrected. Infrared ab-
sorption spectra were measured with a Shimadzu FTIR-8600
spectrophotometer. 1H NMR spectra were measured with a
Bruker AC-300 spectrometer; δs are expressed in parts per million
relative to CHCl3 (7.26 ppm) as an internal reference. MALDI-
TOF MS were recorded on a Thermoquest Finnigan Lasermat
spectrometer; samples were dissolved in CHCl3 and 2,5-dihydrox-
ybenzoic acid or α-cyano-4-hydroxycinnamic acid was used as
the matrix. The elementary analyses were performed at the Mi-
croanalysis Center of Kyoto University.
General Synthetic Procedures. Coupling: Fmoc–AAn–OH
(3.0 mmol), H–AAnꢀ1–…–AA1–OBzl(OC18)3 (1.5 mmol), and
HOBt (3.0 mmol) were dissolved in 60 mL of dry CH2Cl2 under
N2. EDC•HCl (3.3 mmol) and Et3N (0.3 mL) were added to an
ice-chilled solution and the mixture was stirred at room tempera-
ture. After the disappearance of H–AAnꢀ1–…–AA1–OBzl(OC18)3
We examined the preparation of a potential combinatorial
peptide library by using Bzl(OC18)3 group and gel filtration. A
mixture of Fmoc–Ala–OH (0.5 mol amt.) and Fmoc–Phe–OH
(0.5 mol amt.) was coupled with H–Ala–OBzl(OC18)3 (1 mol
amt.) by EDC–HOBt in dichloromethane. The reaction mix-
ture was directly purified by LH-20 gel-filtration and the void
fraction was a 1:1 mixture of Fmoc–Ala–Ala–OBzl(OC18)3
and Fmoc–Phe–Ala–OBzl(OC18)3. The purification procedure
is so simple that the methodology should provide a new strate-

736 Bull. Chem. Soc. Jpn., 74, No. 4 (2001)
Novel Protecting Group for Peptide Library
was checked by TLC (about 1 day), the mixture was washed with
brine six times and dried over Na2SO4. The solvent was evaporat-
ed and a(n) (oily) residue was solidified from methanol to give
crude white Fmoc–AAn–…–AA1–OBzl(OC18)3. In the case of
n ꢁ 1, HO–Bzl(OC18)3 was not completely consumed, even after
3-days reaction, and was removed by washing the crude product
with hexane. Further purification by flash column chromatogra-
phy (FCC) with silica gel (Merck, Kieselgel 60, 9385) and recrys-
tallization afforded the pure desired coupled compound.
Fmoc-Deprotection: To an ice-chilled dry CH2Cl2 solution
(5 mL) of Fmoc–AAn–…–AA1–OBzl(OC18)3 (0.1 mmol) was
added dropwise an ice-chilled dry CH2Cl2 solution (5 mL) of pip-
eridine (3 mL) under N2. The solution was stirred at room temper-
ature for 40 min and ice-chilled methanol (40 mL) was added
dropwise with cooling in an ice–water bath. The resulting white
precipitate was filtered and washed with methanol to give pure H–
AAn–…–AA1–OBzl(OC18)3.
brown products. The filtrate was concentrated in vacuo and the
residue was recrystallized from methanol to give crude 1 as a pale-
yellow powder (24.3 g). Further purification by FCC with CH2Cl2
afforded pure 1; white powder; mp 63–64 ˚C; IR (KBr) 1717
1
(CꢁO), 1587 (CꢁC), 860 cmꢀ1 (C2,6–H); H NMR (CDCl3) δ
0.87 (9H, t, J ꢁ 7 Hz, 3,4,5-O–C17–CH3), 1.25 (84H, m, 3,4,5-O–
C3–(CH2)14), 1.47 (6H, m, 3,4,5-O–C2–CH2), 1.81 (6H, m, 3,4,5-
O–C–CH2), 3.88 (3H, s, 1-COOCH3), 4.00 (6H, t, J ꢁ 6 Hz, 3,4,5-
OCH2), 7.23 (2H, s, 2,6-H). TOFMS found: m/z 943. Calcd for
C62H117O5: MHꢃ, 942. Found: C; 78.81, H; 12.69%. Calcd for
C62H116O5: C; 79.09, H; 12.42%.
3,4,5-Tris(octadecyloxy)benzylAlcohol(HO–Bzl(OC18)3,2).
To an ice-chilled dry Et2O solution (120 mL) of the above crude 1
(3.9 g, < 4.1 mmol) was added LiAlH4 (0.19 g, 5.0 mmol) under
N2 and the mixture was then refluxed for 2 h. After the mixture
was cooled, water (0.2 mL) and aq 15% NaOH (0.2 mL) were
added dropwise to give precipitates. The filtered solid was treated
with THF and the undissolved products were removed. The fil-
trate was concentrated in vacuo and the residue was recrystallized
from 1-propanol to give pure 2 (3.21 g, 76% for the above two
steps); white powder; mp 67–68 ˚C; IR (KBr) 3540, 3480 (O–H),
1595 (CꢁC), 814 cmꢀ1 (C2,6–H); 1H NMR (CDCl3) δ 0.88 (9H, t,
J ꢁ 7 Hz, 3,4,5-O–C17–CH3), 1.25 (84H, m, 3,4,5-O–C3–(CH2)14),
1.46 (6H, m, 3,4,5-O–C2–CH2), 1.79 (6H, m, 3,4,5-O–C–CH2),
3.93 (2H, t, J = 6.5 Hz, 4-OCH2), 3.97 (4H, t, J = 6.5 Hz, 3,5-
OCH2), 4.59 (2H, s, 1-CH2), 6.56 (2H, s, 2,6-H). TOFMS found:
m/z 913. Calcd for C61H116O4: Mꢃ, 913. Found: C; 79.93, H;
13.07%. Calcd for C61H116O4: C; 80.20, H; 12.80%.
Ester-Cleavage:
Fmoc–AAn–…–AA1–OBzl(OC18)3 (15
µmol) was dissolved in 4 M HCl–AcOEt (10 mL) and the solution
was stirred at room temperature overnight. After evaporation in
vacuo, the residue was a 1:1 mixture of Fmoc–AAn–…–AA1–OH
and HO–Bzl(OC18)3. The white solid was treated with a small
amount of hexane and the insoluble acid was separated from the
hexane-soluble alcohol by filtration. The separated solid was a
pure N-protected peptide. The filtrate was evaporated to give pure
HO–Bzl(OC18)3. In the case of n ꢁ 1 and AA1 ꢁ Ala, the chirality
of the resulting Fmoc–Ala–OH was checked: Fmoc–Ala–OH was
first transformed to H–Ala–OH by treatment with piperidine in
dichloromethane as described above and then the unprotected ala-
nine was analyzed by the chiral HPLC as described below.
Fmoc/Ester-Cleavage: Fmoc–AAn–…–AA1-OBzl(OC18)3
(15 µmol) was dissolved in THF (10 mL) and 5% palladium–char-
coal (10 mg) was added to the solution. The suspension was
stirred at room temperature overnight under a hydrogen atmo-
sphere. After filtration and evaporation in vacuo, the residue was a
1:1 mixture of H–AAn–…–AA1–OH and H–Bzl(OC18)3. In the
case of n ꢁ 1 and AA1 ꢁ Ala, the chirality of the resulting H–Ala–
OH was checked as follows. After treatment of the residue with
water, the aqueous solution was analyzed by a chiral HPLC:
SUMICHIRAL OA-6100, 4.6φ ꢂ 250 mm, Sumika Chemical
Analysis Service, aqueous 1 mM CuSO4, 1.0 mL/min, at room
temperature. The peaks of H–D–Ala–OH and H–L–Ala–OH were
completely resolved and their retention times were 7.7 (D) and
14.7 min (L).
Gel-Filtration: Peptide syntheses were performed at a 60-
times smaller scale (1 mL CH2Cl2 solution) than the coupling pro-
cedures described above. After the reaction, the resulting solution
was loaded onto a 9.1 φ ꢂ 193 mm column of hydroxypropylated
cross-linked dextran gel (Amersham Pharmacia Biotech, Sepha-
dex LH-20) and CH2Cl2 was used as the solvent, eluting at 0.40
mL / min. The desired peptides were eluted at 4.2–5.2 mL of elu-
ent volume as the void fraction.
Methyl 3,4,5-Tris(octadecyloxy)benzoate (1). A freshly dis-
tilled DMF suspension (400 mL) of K2CO3 (39 g, 290 mmol) was
fully purged with N2, to which was added stearyl bromide (30.9 g,
93 mmol). The mixture was heated to 70 ˚C under N2 and then
methyl gallate (5.4 g, 30 mmol; commercially available and also
prepared from gallic acid and methanol in the presence of sulfuric
acid, 81%) was added with stirring. After 5-h heating, the cooled
mixture was poured into ice water (1.5 L) and the resulting precip-
itate was filtered and washed with methanol. The pale-brown sol-
id was dissolved in CH2Cl2 and filtered to remove any undissolved
N-(9-Fluorenylmethyloxycarbonyl)alanine 3,4,5-Tris(octa-
decyloxy)benzyl Ester (Fmoc–Ala–OBzl(OC18)3). Coupling
of Fmoc–Ala–OH with 2 by EDC–HOBt gave crude Fmoc–Ala–
OBzl(OC18)3. Further purification by FCC with CH2Cl2 and re-
crystallization from methanol afforded the pure protected alanine
(63% as isolated yield and 80% based on consumed 2); white
powder; mp 97–99 ˚C; IR (KBr) 3310 (N–H), 1734 (ester CꢁO),
1
1690 (amide CꢁO), 1591 (CꢁC), 822 cmꢀ1 (C2,6–H); H NMR
(CDCl3) δ 0.88 (9H, t, J ꢁ 6 Hz, 3,4,5-O–C17–CH3), 1.25 (87H,
m, 3,4,5-O–C3–(CH2)14, Ala–α-CH3), 1.46 (6H, m, 3,4,5-O–C2–
CH2), 1.79 (6H, m, 3,4,5-O–C–CH2), 3.92 (2H, t, J ꢁ 6 Hz, 4-
OCH2), 3.94 (4H, t, J ꢁ 6 Hz, 3,5-OCH2), 4.23 (1H, t, J ꢁ 7 Hz,
Fmoc–9-H), 4.38 (2H, d, J ꢁ 6 Hz, Fmoc–9-CH2), 4.46 (1H, m,
Ala–α-H), 5.08 (2H, s, 1-CH2), 5.38 (1H, d, J = 8 Hz, Ala–NH),
6.52 (2H, s, 2,6-H), 7.31, 7.40 (each 2H, t, J ꢁ 7 Hz, Fmoc–
2,3,6,7-H), 7.59, 7.76 (each 2H, d, J ꢁ 7 Hz, Fmoc–1,4,5,8-H).
TOFMS found: m/z 1207. Calcd for C79H132NO7: MHꢃ, 1207.
Alanine 3,4,5-Tris(octadecyloxy)benzyl Ester (H–Ala–O–
Bzl(OC18)3). Deprotection of the Fmoc group in Fmoc–Ala–
OBzl(OC18)3 by piperidine gave H–Ala–OBzl(OC18)3 (92%);
white powder; mp 62–65 ˚C; IR (KBr) 1734 (ester CꢁO), 1589
(CꢁC), 818 cmꢀ1 (C2,6–H); 1H NMR (CDCl3) δ 0.88 (9H, t, J ꢁ 6
Hz, 3,4,5-O–C17–CH3), 1.25 (87H, m, 3,4,5-O–C3–(CH2)14, Ala–
α-CH3), 1.46 (6H, m, 3,4,5-O–C2–CH2), 1.79 (6H, m, 3,4,5-O–C–
CH2), 3.59 (1H, q, J = 7 Hz, Ala–α-H), 3.93 (2H, t, J ꢁ 6 Hz, 4-
OCH2), 3.95 (4H, t, J = 6 Hz, 3,5-OCH2), 5.04 (2H, s, 1-CH2),
6.52 (2H, s, 2,6-H).
N-(9-Fluorenylmethyloxycarbonyl)phenylalanylalanine 3,4,
5-Tris(octadecyloxy)benzyl Ester
(Fmoc–Phe–Ala–OBzl–
(OC18)3). Coupling of Fmoc–Phe–OH with H–Ala–OBzl–
(OC18)3 by EDC–HOBt gave crude Fmoc–Phe–Ala–OBzl(OC18)3.
Further purification by FCC with 5% Et2O–CH2Cl2 and recrystal-
lization from methanol afforded the pure protected dipeptide
(67%); white powder; mp 102–104 ˚C; IR (KBr) 3294 (N–H),

H. Tamiaki et al.
Bull. Chem. Soc. Jpn., 74, No. 4 (2001) 737
1736 (ester CꢁO), 1692 (urethane amide CꢁO), 1653 (amide
CꢁO), 1591 (CꢁC), 820 cmꢀ1 (C2,6–H); 1H NMR (CDCl3) δ 0.88
(9H, t, J ꢁ 6 Hz, 3,4,5-O–C17–CH3), 1.25 (87H, m, 3,4,5-O–C3–
(CH2)14, Ala–α-CH3), 1.46 (6H, m, 3,4,5-O–C2-CH2), 1.79 (6H,
m, 3,4,5-O–C–CH2), 3.07 (2H, m, Phe–α-CH2), 3.93 (2H, t, J ꢁ 6
Hz, 4-OCH2), 3.95 (4H, t, J ꢁ 6 Hz, 3,5-OCH2), 4.19 (1H, t, J ꢁ 7
Hz, Fmoc–9-H), 4.33 (1H, m, Ala–α-H), 4.43 (2H, m, Fmoc–9-
CH2), 4.54 (1H, m, Phe–α-H), 5.04 (2H, s, 1-CH2), 5.30 (1H, br-d,
Phe–NH), 6.30 (1H, br-d, Ala–NH), 6.50 (2H, s, 2,6-H), 7.18–
7.25 (5H, m, Phe–β-C6H5), 7.30, 7.40 (each 2H, t, J ꢁ 7 Hz,
Fmoc–2,3,6,7-H), 7.54, 7.76 (each 2H, d, J ꢁ 7 Hz, Fmoc–
1,4,5,8-H). TOFMS found: m/z 1377. Calcd for C88H140N2NaO8:
MNaꢃ, 1377.
1,4,5,8-H). TOFMS found: m/z 1490. Calcd for C94H151N3NaO9:
MNaꢃ, 1490.
3,4,5-Tris(octadecyloxy)benzyl Chloride (Cl–Bzl(OC18)3, 3).
Following the reported procedure,8 reaction of 2 with SOCl2 in the
presence of a catalytic amount of DMF in dry CH2Cl2 and recrys-
tallization from methanol afforded pure 3 (92%); white powder;
1
mp 65–66 ˚C; IR (KBr) 1593 (CꢁC), 833 cmꢀ1 (C2,6–H); H
NMR (CDCl3) δ 0.88 (9H, t, J = 7 Hz, 3,4,5-O–C17–CH3), 1.25
(84H, m, 3,4,5-O–C3–(CH2)14), 1.46 (6H, m, 3,4,5-O–C2–CH2),
1.79 (6H, m, 3,4,5-O–C–CH2), 3.93 (2H, t, J ꢁ 6.5 Hz, 4-OCH2),
3.96 (4H, t, J ꢁ 6.5 Hz, 3,5-OCH2), 4.51 (2H, s, 1-CH2), 6.56 (2H,
s, 2,6-H). TOFMS found: m/z 932. Calcd for C61H11635ClO3: MHꢃ,
932. Found: C; 78.69, H; 12.51%. Calcd for C61H115ClO3: C;
78.61, H; 12.44%.
N-(9-Fluorenylmethyloxycarbonyl)alanylalanine 3,4,5-Tris
(octadecyloxy)benzyl Ester (Fmoc–Ala1–Ala2–OBzl(OC18)3).
Coupling of Fmoc–Ala–OH with H–Ala–OBzl(OC18)3 by EDC–
HOBt gave crude Fmoc–Ala–Ala–OBzl(OC18)3. Further purifica-
tion by FCC with 5% Et2O–CH2Cl2 and recrystallization from
methanol afforded the pure protected dipeptide (70%); white pow-
der; mp 88–92 ˚C; IR (KBr) 3294 (N–H), 1732 (ester CꢁO), 1693
(urethane amide CꢁO), 1651 (amide CꢁO), 1589 (CꢁC), 820
′ ′ ′
3,4,5-Tris[3 ,4 ,5 -tris(octadecyloxy)benzyloxy]benzyl Alco-
hol (HO–Bzl[OBzl(OC18)3]3, 4). Similarly to the synthesis of 1,
a reaction of methyl gallate and 3 at 74 ˚C for 4 h afforded crude
methyl 3,4,5-tris[3′,4′,5′-tris(octadecyloxy)benzyloxy]benzoate.
Further purification by FCC with 30% hexane–CH2Cl2 gave the
pure ester (95%); white powder; 1H NMR (CDCl3) δ 0.88 (27H, t,
J ꢁ 6 Hz, 3′,4′,5′-O–C17–CH3), 1.25 (252H, m, 3′,4′,5′-O–C3–
(CH2)14), 1.44 (18H, m, 3′,4′,5′-O–C2–CH2), 1.72 (18H, m,
3′,4′,5′-O–C–CH2), 3.75 (4H, t, J ꢁ 6.5 Hz, 3′,5′-OCH2 on the 4-
position), 3.87 (8H, t, J ꢁ 5 Hz, 3′,5′-OCH2 on the 3,5-positions),
3.87 (3H, s, 1-COOCH3), 3.97 (6H, m, 4′-OCH2), 5.02 (6H, s,
3,4,5-OCH2), 6.59 (2H, s, 2′,6′-H on the 4-position), 6.62 (4H, s,
2′,6′-H on the 3,5-position), 7.37 (2H, s, 2,6-H).
Similarly to the synthesis of 2, the reduction of the above crude
ester with LiAlH4 afforded 4 (90% for the two steps); white pow-
der; mp 75–77 ˚C; IR (KBr) 1589 (CꢁC), 814 cmꢀ1 (C2,6,2′,6′-H);
1H NMR (CDCl3) δ 0.88 (27H, t, J ꢁ 6 Hz, 3′,4′,5′-O–C17–CH3),
1.25 (252H, m, 3′,4′,5′-O–C3–(CH2)14), 1.44 (18H, m, 3′,4′,5′-O–
C2–CH2), 1.72 (18H, m, 3′,4′,5′-O–C–CH2), 3.75 (4H, t, J ꢁ 6.5
Hz, 3′,5′-OCH2 on the 4-position), 3.87 (8H, t, J ꢁ 5 Hz, 3′,5′-
OCH2 on the 3,5-positions), 3.97 (6H, m, 4′-OCH2), 4.58 (2H, s,
1-CH2), 5.02 (6H, s, 3,4,5-OCH2), 6.56 (2H, s, 2,6-H), 6.59 (2H, s,
2′,6′-H on the 4-position), 6.62 (4H, s, 2′,6′-H on the 3,5-posi-
tion). Found: C; 79.43, H; 12.77%. Calcd for C190H350O13•H2O: C;
79.77, H; 12.40%.
1
cmꢀ1 (C2,6–H); H NMR (CDCl3) δ 0.87 (9H, t, J ꢁ 6 Hz, 3,4,5-
O–C17-CH3), 1.25 (90H, m, 3,4,5-O–C3–(CH2)14, Ala1,2–α-CH3),
1.45 (6H, m, 3,4,5-O–C2-CH2), 1.77 (6H, m, 3,4,5-O–C–CH2),
3.93 (2H, t, J ꢁ 6 Hz, 4-OCH2), 3.95 (4H, t, J ꢁ 6 Hz, 3,5-OCH2),
4.22 (1H, t, J ꢁ 7 Hz, Fmoc–9-H), 4.24 (1H, m, Ala2–α-H), 4.40
(2H, d, J ꢁ 7 Hz, Fmoc–9-CH2), 4.60 (1H, m, Ala1α-H), 5.06
(2H, s, 1-CH2), 5.37 (1H, br, Ala1–NH), 6.47 (1H, br, Ala2–NH),
6.51 (2H, s, 2,6-H), 7.31, 7.40 (each 2H, t, J ꢁ 7 Hz, Fmoc–
2,3,6,7-H), 7.58, 7.76 (each 2H, d, J ꢁ 7 Hz, Fmoc–1,4,5,8-H).
Phenylalanylalanine 3,4,5-Tris(octadecyloxy)benzyl Ester
(H–Phe–Ala–OBzl(OC18)3). Deprotection of Fmoc group in
Fmoc–Phe–Ala–OBzl(OC18)3 by piperidine gave H–Phe–Ala–
OBzl(OC18)3 (92%); white powder; 1H NMR (CDCl3) δ 0.88 (9H,
t, J ꢁ 6 Hz, 3,4,5-O–C17–CH3), 1.25 (87H, m, 3,4,5-O–C3–
(CH2)14, Ala–α-CH3), 1.46 (6H, m, 3,4,5-O–C2–CH2), 1.78 (6H,
m, 3,4,5-O–C–CH2), 2.71 (1H, dd, J ꢁ 9, 14 Hz, Phe–α-CH), 3.25
(1H, dd, J ꢁ 4, 14 Hz, Phe–α-CH), 3.63 (1H, dd, J ꢁ 4, 9 Hz,
Phe–α-H), 3.93 (2H, t, J ꢁ 6 Hz, 4-OCH2), 3.96 (4H, t, J ꢁ 6 Hz,
3,5-OCH2), 4.62 (1H, quintet, J ꢁ 7.5 Hz, Ala–α-H), 5.06 (2H, s,
1-CH2), 6.52 (2H, s, 2,6-H), 7.20–7.34 (5H, m, Phe–β-C6H5), 7.79
(1H, d, J ꢁ 7.5 Hz, Ala–NH).
′ ′
N-(9-Fluorenylmethyloxycarbonyl)alanine 3,4,5-Tris[3 ,4 ,
′
5 -tris(octadecyloxy)benzyloxy]benzyl Ester (Fmoc–Ala–O–
Bzl[OBzl(OC18)3]3). Coupling of Fmoc–Ala–OH with 4 by
EDC–HOBt gave crude Fmoc–Ala–OBzl[OBzl(OC18)3]3. Further
purification by FCC with CH2Cl2 and recrystallization from meth-
anol afforded the pure protected alanine (30%); white powder; 1H
NMR (CDCl3) δ 0.88 (27H, t, J = 6 Hz, 3′,4′,5′-O–C17–CH3), 1.25
(255H, m, 3′,4′,5′-O–C3–(CH2)14, Ala–α-CH3), 1.44 (18H, m,
3′,4′,5′-O–C2–CH2), 1.72 (18H, m, 3′,4′,5′-O–C–CH2), 3.75 (4H,
t, J ꢁ 6.5 Hz, 3′,5′-OCH2 on the 4-position), 3.87 (8H, t, J ꢁ 5
Hz, 3′,5′-OCH2 on the 3,5-positions), 3.97 (6H, m, 4′-OCH2), 4.23
(1H, t, J ꢁ 7 Hz, Fmoc–9-H), 4.38 (2H, d, J ꢁ 6 Hz, Fmoc–9-
CH2), 4.46 (1H, m, Ala–α-H), 5.02 (6H, s, 3,4,5-OCH2), 5.08 (2H,
s, 1-CH2), 5.38 (1H, d, J ꢁ 8 Hz, Ala–NH), 6.52 (2H, s, 2,6-H),
6.59 (2H, s, 2′,6′-H on the 4-position), 6.62 (4H, s, 2′,6′-H on the
3,5-position), 7.31, 7.40 (each 2H, t, J ꢁ 7 Hz, Fmoc–2,3,6,7-H),
7.59, 7.76 (each 2H, d, J ꢁ 7 Hz, Fmoc–1,4,5,8-H).
N-(9-Fluorenylmethyloxycarbonyl)leucylphenylalanylala-
nine 3,4,5-Tris(octadecyloxy)benzyl Ester (Fmoc–Leu–Phe–
Ala–OBzl(OC18)3). Coupling of Fmoc–Leu–OH with H–Phe–
Ala–OBzl(OC18)3 by EDC–HOBt gave crude Fmoc–Leu–Phe–
Ala–OBzl(OC18)3. Further purification by FCC with 15% Et2O–
CH2Cl2 and recrystallization from methanol afforded the pure pro-
tected tripeptide (52%); white powder; mp 147–149 ˚C; IR (KBr)
3279 (N–H), 1740 (ester CꢁO), 1693 (urethane amide CꢁO),
1
1647 (amide CꢁO), 1591 (CꢁC), 818 cmꢀ1 (C2,6–H); H NMR
(CDCl3) δ 0.88 (15H, t, J ꢁ 6 Hz, 3,4,5-O–C17–CH3, Leu–γ-CH3),
1.25 (87H, m, 3,4,5-O–C3–(CH2)14, Ala–α-CH3), 1.46 (6H, m,
3,4,5-O–C2–CH2), 1.57 (3H, m, Leu–α-CH2, γ-H), 1.79 (6H, m,
3,4,5-O–C–CH2), 3.07 (2H, d, J ꢁ 6 Hz, Phe–α-CH2), 3.93 (2H, t,
J ꢁ 6 Hz, 4-OCH2), 3.95 (4H, t, J ꢁ 6 Hz, 3,5-OCH2), 4.12 (1H,
m, Leu–α-H), 4.19 (1H, t, J ꢁ 7 Hz, Fmoc–9-H), 4.34 (1H, m,
Ala–α-H), 4.42–4.53 (2H, m, Fmoc–9-CH2), 4.64 (1H, q, J ꢁ 7
Hz, Phe–α-H), 5.03 (2H, s, 1-CH2), 5.05 (1H, br, Leu–NH), 6.46,
6.55 (each 1H, d, J ꢁ 6 Hz, Ala, Phe–NH), 6.51 (2H, s, 2,6-H),
7.16–7.23 (5H, m, Phe–β-C6H5), 7.31, 7.41 (each 2H, t, J ꢁ 7 Hz,
Fmoc–2,3,6,7-H), 7.57, 7.77 (each 2H, d, J ꢁ 7 Hz, Fmoc–
3,4,5-Tris(octadecyloxy)benzoic Acid. To an H2O–EtOH
(1:3) solution (133 mL) of methyl ester 1 (12.52 g) was added
KOH (7.53 g). The mixture was refluxed for 4 h, and then left
standing at room temperature overnight. The precipitated solid
was separated by decantation, and was stirred vigorously for sev-
eral days in 0.6 M HCl (250 mL) and CH2Cl2 (150 mL). After

738 Bull. Chem. Soc. Jpn., 74, No. 4 (2001)
Novel Protecting Group for Peptide Library
evaporation of CH2Cl2, the residue was filtered, washed with wa-
ter and methanol, and dried to give the crude acid. Recrystalliza-
tion from 1-propanol (400 mL), washing with methanol and dry-
ing over P2O5 gave the pure carboxylic acid (11.01 g, 89%); white
powder; mp 86–87 ˚C; IR (KBr) 1682 (CꢁO), 1587 (CꢁC), 864
HPLC analyses.
References
1
cmꢀ1 (C2,6–H); H NMR (CDCl3) δ 0.88 (9H, t, J ꢁ 6 Hz, 3,4,5-
1
F. Balkenhohl, C. von dem Bussche-Hünnefeld, A. Lansky,
and C. Zechel, Angew. Chem., Int. Ed. Engl., 35, 2288 (1996); R.
A. Houghten, C. Pinilla, J. R. Appel, S. E. Blondelle, C. T. Dooley,
J. Eichler, A. Nefzi, and J. M. Ostresh, J. Med. Chem., 42, 3743
(1999).
O–C17–CH3), 1.25 (84H, m, 3,4,5-O–C3–(CH2)14), 1.47 (6H, m,
3,4,5-O–C2–CH2), 1.79 (6H, m, 3,4,5-O–C–CH2), 4.01 (4H, t, J ꢁ
6 Hz, 3,5-OCH2), 4.03 (2H, t, J ꢁ 6 Hz, 4-OCH2), 7.29 (2H, s,
2,6-H). Found: C; 78.70, H; 12.54%. Calcd for C61H114O5: C;
78.99, H; 12.39%.
2
S. R. Whaley, D. S. English, E. L. Hu., P. F. Barbara, and A.
M. Belcher, Nature, 405, 665 (2000).
N. K. Terrett, M. Gardner, D. W. Gordon, R. J. Kobylecki,
and J. Steele, Tetrahedron, 51, 8135 (1995).
3,4,5-Tris(octadecyloxy)benzaldehyde. To a dry CH2Cl2 so-
lution (80 mL) of substituted benzyl alcohol 2 (2.0 g, 2.2 mmol)
was added pyridinium chlorochromate (0.71 g, 3.3 mmol) and the
reaction mixture was stirred at room temperature for 3 h. After
evaporation, the residue was treated with Et2O and the undis-
solved materials were removed. The filtrate was concentrated in
vacuo and the residue was purified by FCC with CH2Cl2 to give
the pure aldehyde (1.78 g, 87%); white powder; mp 75–76 ˚C; IR
(KBr) 2731 (OC–H), 1693 (CꢁO), 1585 (CꢁC), 824 cmꢀ1 (C2,6–
H); 1H NMR (CDCl3) δ 0.88 (9H, t, J ꢁ 7 Hz, 3,4,5-O–C17–CH3),
1.25 (84H, m, 3,4,5-O–C3–(CH2)14), 1.46 (6H, m, 3,4,5-O–C2–
CH2), 1.79 (6H, m, 3,4,5-O–C–CH2), 4.03 (4H, t, J ꢁ 6 Hz, 3,5-
OCH2), 4.05 (2H, t, J ꢁ 6 Hz, 4-OCH2), 7.08 (2H, s, 2,6-H), 9.82
(1H, s, CHO). TOFMS found: m/z 912. Calcd for C61H115O4:
MHꢃ, 912. Found: C; 79.97, H; 12.79%. Calcd for C61H114O4: C;
80.37, H; 12.61%.
3
4
5
D. J. Gravert and K. D. Janda, Chem. Rev., 97, 489 (1997).
R. M. Kim, M. Manna, S. M. Hutchins, P. R. Griffin, N. A.
Yates, A. M. Bernick, and K. T. Chapman, Proc. Natl. Acad. Sci.
U.S.A., 93, 10012 (1996).
6
Preliminary report: H. Tamiaki, T. Obata, and K. Toma, in
“Peptide Science 1998,” ed by M. Kondo, Protein Research Foun-
dation, Osaka (1999), pp. 125–128.
7
M. Bodanszky, “Peptide Chemistry,” 2nd ed, Springer-Ver-
lag, New York (1993).
8
V. S. K. Balagurusamy, G. Ungar, V. Percec, and G.
Johansson, J. Am. Chem. Soc., 119, 1539 (1997).
9
Dissolution of Fmoc–Ala–OBzl(OC18)3 in trifluoroacetic
acid (TFA) and successive stirring at room temperature for 4 h in-
duced partial cleavage of the benzyl ester bond (< 10%), showing
the treatment of TFA was ineffective for the cleavage.
10 For example, see “The Combinatorial Chemistry Catalog,”
Novabiochem (http://www.nova.ch).
We thank Mr. Atsushi Tanaka of Asahi Chemical Industry, Co.,
Ltd. for his measurements of MALDI-TOF MS and Mr. Koji
Kumon of Ritsumeikan University for his assistance with chiral