Cyclohexyl ether as a new hydroxy-protecting group for serine and threonine in peptide synthesis

Article abstract of DOI:10.1039/b001261k

A new hydroxy-protecting group for Ser and Thr, cyclohexyl (Chx), has been developed, and its application to peptide synthesis is described. The Chx group is introduced to the hydroxy functions of Boc-Ser-OH and Boc-Thr-OH in two steps; Boc-Ser-OH and Boc-Thr-OH are treated with Nail and then allowed to react with 3-bromocyclohexene to afford N-Boc-O-(cyclohex-2-enyl)-Ser and N-Boc-O-(cyclohex-2-enyl)-Thr in satisfactory yields, which are hydrogenated in the presence of PtO₂ to give Boc-Ser(Chx)-OH and Boc-Thr(Chx)-OH in good yields. The O-Chx group is stable to various acidic and basic conditions including TFA and 20% piperidine in DMF. It is not removed with catalytic hydrogenation over Pd-charcoal. The Chx group is, however, removed quantitatively with 1 mol dm^-3 trifluoromethanesulfonic acid-thioanisole in TFA over a short period. These results indicate that the Chx group is suitable for the hydroxy-protection of Ser and Thr in peptide synthesis based on Boc-chemistry and can be used also in combination with either N^α-Fmoc or N^α-Z-protection. The apparent rate constant for removal of the Chx group with 50% TFA in CH₂Cl₂ (k_Chx) is found to be less than a twentieth of that of the Bzl group (k_Bzl), confirming the substantial stability of the Chx group under common Boc-deprotection conditions. Simulations of solid-phase peptide syntheses using k_Chx and k_Bzl indicate that the Chx group would be adequate for solid-phase synthesis of large peptides and proteins. Solid-phase synthesis of a peptide and conventional solution synthesis of a protected peptide segment using Chx protection are also demonstrated.

Full text of DOI:10.1039/b001261k

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Cyclohexyl ether as a new hydroxy-protecting group for serine and
threonine in peptide synthesis†,1
Yasuhiro Nishiyama,*,‡ Suguru Shikama, Ken-ichi Morita and Keisuke Kurita
1
Department of Industrial Chemistry, Faculty of Engineering, Seikei University, Musashino-shi,
Tokyo 180-8633, Japan
Received (in Cambridge, UK) 15th February 2000, Accepted 14th April 2000
Published on the Web 18th May 2000
A new hydroxy-protecting group for Ser and Thr, cyclohexyl (Chx), has been developed, and its application
to peptide synthesis is described. The Chx group is introduced to the hydroxy functions of Boc-Ser-OH and
Boc-Thr-OH in two steps; Boc-Ser-OH and Boc-Thr-OH are treated with NaH and then allowed to react with
3-bromocyclohexene to afford N-Boc-O-(cyclohex-2-enyl)-Ser and N-Boc-O-(cyclohex-2-enyl)-Thr in satisfactory
yields, which are hydrogenated in the presence of PtO2 to give Boc-Ser(Chx)-OH and Boc-Thr(Chx)-OH in good
yields. The O-Chx group is stable to various acidic and basic conditions including TFA and 20% piperidine in DMF.
It is not removed with catalytic hydrogenation over Pd–charcoal. The Chx group is, however, removed quantitatively
with 1 mol dmϪ3 trifluoromethanesulfonic acid–thioanisole in TFA over a short period. These results indicate that
the Chx group is suitable for the hydroxy-protection of Ser and Thr in peptide synthesis based on Boc-chemistry and
can be used also in combination with either Nα-Fmoc or Nα-Z-protection. The apparent rate constant for removal
of the Chx group with 50% TFA in CH2Cl2 (kChx) is found to be less than a twentieth of that of the Bzl group (kBzl),
confirming the substantial stability of the Chx group under common Boc-deprotection conditions. Simulations of
solid-phase peptide syntheses using kChx and kBzl indicate that the Chx group would be adequate for solid-phase
synthesis of large peptides and proteins. Solid-phase synthesis of a peptide and conventional solution synthesis
of a protected peptide segment using Chx protection are also demonstrated.
(TFMSA)–thioanisole in TFA,7 etc. The Bzl group is, however,
Introduction
partially lost during the treatment with 50% TFA in CH2Cl2.§
Protection of side-chain functional groups of trifunctional
The hydroxy function regenerated by losing the Bzl group at
amino acids has been a major consideration in peptide chem-
every Nα-deprotection step would be acylated at the subsequent
istry. A multitude of protecting groups and refined systems for
coupling steps to give a variety of O-branched peptides, which
their removal have been developed, and currently the tactics of
may also undergo β-elimination with production of dehydro-
side-chain protection are an almost routine procedure in the
alanine peptides.6 Particularly in SPPS of large peptides or
solid-phase synthesis of simple peptides.2 However, synthesis of
proteins, accumulation of side products would severely compli-
large peptides or proteins by solid-phase and conventional solu-
cate the isolation of the desired product, even though the
tion methods requires more stringent or absolute selectivity in
hydroxy-protecting group is lost only in a small amount. The
removal of α-amino-protecting groups and side-chain protect-
Bzl group is, therefore, not entirely adequate for SPPS of long
peptides and proteins. More TFA-stable hydroxy-protecting
groups have been thus required for efficient peptide synthesis,
and developed mainly by introducing electron-withdrawing
ing groups. Preparation of combinatorial peptide libraries3 also
seems to require the strict selectivity of protective-group cleav-
age, since the resulting libraries are usually screened for various
functions including biological activity without purification
and intensive characterization. Protecting groups with unique
substituents on the phenyl ring of the benzyl moiety, such as
bromo8 and N,N-dimethylcarbamoyl.9,¶
stability/removability profiles have opened new synthetic strat-
Alternatively, sec-alkyl skeletons, such as cyclohexyl (Chx),
egies for complex peptides, such as multiple antigenic peptides
have been known to be suitable as highly TFA-stable protecting
having two distinct epitopes,4 and regioselectively sulfonated
groups.10–13 For example, an apparent rate constant in decom-
Tyr-containing peptides.5 Thus the development of new pro-
position of Asp(OChx) in 50% TFA in CH2Cl2 was reported to
tecting groups remains a major challenge in peptide chemistry.
be less than a hundredth of that of Asp(OBzl).11 A Chx group
The hydroxy functions of Ser and Thr are usually masked to
on the hydroxy function of Tyr was also much more stable to
prevent the unwanted O-acylation.6 The most common protect-
ing group for Ser and Thr in solid-phase peptide synthesis
TFA than a Bzl group.12 The Chx protection of these amino
acids can be smoothly removed with anhydrous HF or 1 mol
(SPPS) based on the Boc-chemistry is benzyl (Bzl), which can
dmϪ3 TFMSA–thioanisole in TFA in a short period. These
be readily deprotected by hydrogenation over Pd or acidolysis
features have prompted us to apply the Chx group to the
side-chain protection of Ser and Thr in peptide synthesis. This
with anhydrous HF, 1 mol dmϪ3 trifluoromethanesulfonic acid
paper deals with the synthesis of Boc-Ser(Chx)-OH and Boc-
Thr(Chx)-OH (Fig. 1), stability and removability of the O-Chx
group under various conditions, and its application to SPPS
and conventional peptide synthesis in solution.
† Amino acids used in this study are of -configuration. Abbreviations
used in this report for amino acids, peptides, and their derivatives are
those recommended by the IUPAC–IUB Joint Commission on Bio-
chemical Nomenclature: Biochem. J., 1984, 219, 345; Eur. J. Biochem.,
1984, 138, 9; Pure Appl. Chem., 1984, 56, 595.
‡ Present address: Department of Pathology and Laboratory Medicine,
The University of Texas-Houston Medical School, 6431 Fannin, MSB
2.252, Houston, Texas 77030, U.S.A.
§ 3% (Ser) and 5% (Thr) loss after 23 h treatment.8
¶ For instance, only 1.3% of 4-bromobenzyl was reported to be lost on
treatment with 50% TFA in CH2Cl2 for 71 h.8
DOI: 10.1039/b001261k
J. Chem. Soc., Perkin Trans. 1, 2000, 1949–1954
This journal is © The Royal Society of Chemistry 2000
1949

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Fig. 1 Boc-Ser(Chx)-OH (R = H) and Boc-Thr(Chx)-OH (R = Me).
Results and discussion
Synthesis of Boc-Ser(Chx)-OH and Boc-Thr(Chx)-OH
Although Boc-Ser(Bzl)-OH can be readily prepared from Boc-
Ser-OH and benzyl bromide by the Williamson synthesis,14
a
similar reaction using cyclohexyl bromide instead of benzyl
bromide failed to give Boc-Ser(Chx)-OH. This failure would be
a result of the insufficient reactivity of the sec-alkyl halide in
nucleophilic substitution. Thus we tried the two-step synthesis
of Boc-Ser(Chx)-OH, as shown in Scheme 1, involving the
Fig.
2
Best-fit lines for apparent first-order-deprotection of
Ser(Chx) in comparison with Ser(Bzl), Lys(Z), Lys(2-ClZ), Tyr(Bzl)
and Tyr(Cl2Bzl). 2-ClZ, 2-chlorobenzyloxycarbonyl; Cl2Bzl, 2,6-
dichlorobenzyl.
The stability of the Chx group under various basic condi-
tions was examined using Boc-Ser(Chx)-OH by similar pro-
cedures to those for acid-stability tests. As shown in Table 1, the
Chx group was not removed with 10% Et3N in DMF, 10% aq.
NaHCO3, 2 mol dmϪ3 aq. NaOH, and 20% piperidine in DMF.
These results indicate that the Chx group is satisfactorily stable
under various basic conditions including those for saponifi-
cation of esters and for β-elimination of fluoren-9-ylmethyl-
based protecting groups.
Boc-Ser(Chx)-OH was exposed to catalytic hydrogenolytic
conditions, by which the Bzl and 4-bromobenzyl groups were
removed quantitatively. Removal of the Chx group was not
detectable.
From these results, the Chx group appears suitable for the
hydroxy-protection for Ser and Thr in peptide synthesis based
on the Boc-chemistry. In addition, the O-Chx group can be
used in combination with Nα-Fmoc and Nα-Z protections, if
desired. Furthermore, the cyclohexyl ether has a unique
stability/removability profile different from other major
hydroxy-protecting groups, such as But, Bzl, and fluoren-9-yl-
methyl, and thus they are removable individually without
significant loss of the Chx group.
Scheme 1 Reagents: (i) NaH; (ii) 3-bromocyclohexene; (iii) H2, PtO2.
introduction of the cyclohex-2-enyl (Che) moiety and its
hydrogenation to Chx, since the Che ether can be deduced to be
synthesized readily by the nucleophilic substitution reaction
of 3-bromocyclohexene (Che-Br), which has a highly reactive
allyl-halide-like structure, with the sodium alkoxide derived
from Boc-Ser-OH. As expected, Boc-Ser(Che)-OH could be
obtained in a satisfactory yield. Hydrogenation of Boc-
Ser(Che)-OH over Pd-charcoal failed to give Boc-Ser(Chx)-
OH.|| Boc-Ser(Che)-OH could, however, be hydrogenated over
PtO2 to afford Boc-Ser(Chx)-OH in good yield. Although this
route requires an additional hydrogenation step, the overall
yield of Boc-Ser(Chx)-OH from Boc-Ser-OH was comparable
to that of Boc-Ser(Bzl)-OH.14
Boc-Thr(Chx)-OH could be synthesized in a similar manner,
and the overall yield of Boc-Thr(Chx)-OH from Boc-Thr-OH
was higher than that of Boc-Thr(Bzl)-OH.14
Kinetics of Chx loss in 50% TFA in CH2Cl2
Stability/removability profile of the O-Chx group
To compare the acid stability of Chx and Bzl in detail, we exam-
ined the kinetics of Chx loss under common Boc-deprotecting
conditions.
The acid stability of the O-Chx group was tested using Fmoc-
Ser(Chx)-OH, which was synthesized from Boc-Ser(Chx)-OH
in the usual manner. Fmoc-Ser(Chx)-OH was treated with
TFA, 4 mol dmϪ3 HCl in AcOEt, 1 mol dmϪ3 trimethylsilyl
bromide (TMSBr)–thioanisole in TFA,15 and 1 mol dmϪ3
TFMSA–thioanisole in TFA, and then portions of the test
solutions were subjected to HPLC to determine the amounts of
survived Fmoc-Ser(Chx)-OH. No appreciable removal of the
Chx group was observed in the treatment with TFA and 4 mol
dmϪ3 HCl in AcOEt as included in Table 1. Furthermore, the
Chx group was stable to 1 mol dmϪ3 TMSBr–thioanisole in
TFA, by which the Bzl and Z groups were removed quanti-
tatively. These results clearly indicate the substantial acid stabil-
ity of the Chx group. The Chx group was, however, removed
quantitatively with 1 mol dmϪ3 TFMSA–thioanisole in TFA at
rt within 30 min.
Fmoc-Ser(Chx)-OH was dissolved in 50% TFA in CH2Cl2,
and the amount of survived Fmoc-Ser(Chx)-OH was deter-
mined with HPLC after 2, 6, 24, 48, 96, and 192 h. Only 3.9%
loss was observed after 192 h, revealing the excellent stability
of the Chx group. The apparent first-order rate constant for
removal of the Chx group with 50% TFA in CH2Cl2 (kChx) was
determined from the slope of the best-fit line through data
points plotted on a grid of ln(X0/Xt) [where X0 is the initial
concentration of Fmoc-Ser(Chx)-OH and Xt is the concen-
tration of Fmoc-Ser(Chx)-OH at a given time t] vs. time (Fig. 2)
based on the rate law, kChxؒt = ln(X0/Xt). The kChx was deter-
mined as 0.46 × 10Ϫ8 sϪ1. Previously, the apparent rate constant
of Bzl loss under similar conditions was determined as 10.7 ×
10Ϫ8 sϪ1 16
These values allowed calculation of the amounts
.
of Chx and Bzl to be removed in a single standard Nα-Boc-
deprotection (rt, 20 min) as 0.00055 and 0.013%, respectively.
This indicates that the Chx group withstands Boc-deprotection
conditions considerably better than Bzl does.
|| Boc-Ser(Che)-OH was recovered with a small amount of Boc-Ser-
OH, which was regenerated possibly by undesirable hydrogenolytic
cleavage of the Che ether bond.
1950
J. Chem. Soc., Perkin Trans. 1, 2000, 1949–1954

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Table 1 Stability and removability of the Chx group
% R-Ser(Chx)-OH surviveda
Conditions
0.5
1
2
4
8
24 h
TFA, rt
100
100
100
100
100
100
100
100
4 mol dmϪ3 HCl in AcOEt, rt
1 mol dmϪ3 TMSBr–thioanisole in TFA,
ice-bath temperature
100
0
98
1 mol dmϪ3 TFMSA–thioanisole in TFA, rt
10% aq. NaHCO3, rt
10% Et3N in DMF, rt
20% piperidine in DMF, rt
2 mol dmϪ3 aq. NaOH, rt
H2/Pd–C in MeOH–water
100
100
100
100
100
100
100
100
100
100
100
100
100
a In acid treatments, R = Fmoc. In other, R = Boc.
Table 2 Calculated yields (%) of peptides that maintain complete protection of Ser residues for SPPS of three peptides from the human immuno-
globulin Eu
Variable region of
light chain (108
amino acid
residues)
16
774
Complete light
chain (214 amino
acid residues)
Complete heavy
chain (440 amino
acid residues)
Number of Ser residues
Cyclesa
32
3389
53
11 223
Protection of Ser
Calculated yield (%)b
Chx
Bzl
99.6
98.1
94.0
90.5c
64.7c
23.7c
a Sum of the TFA exposures of the individual Ser residues, where the TFA exposure of the residue at position P is P Ϫ 1 cycles. b Mole % of the
peptides having all Ser residues still protected upon complete assembly of the resin-bound peptide; based on deprotection by 50% TFA in CH2Cl2 for
20 min cycleϪ1 c Ref. 16.
.
Application of Boc-Ser(Chx)-OH to SPPS
To demonstrate the applicability of the Chx group to practical
Boc-SPPS,
a simple hexapeptide, Ac-Tyr-Ile-Gly-Ser-Arg-
βAla-OH (βAla: β-alanine), which is an analogue of laminin-
related antimetastatic peptide YIGSR,18 was synthesized.
The desired sequence was constructed on the Merrifield resin19
bearing Boc-βAla by the successive couplings of Boc-Arg(Tos)-
OH20 (Tos: p-tolylsulfonyl), Boc-Ser(Chx)-OH, Boc-Gly-
OH, Boc-Ile-OH, and Boc-Tyr(Cl2Bzl)-OH16 (Cl2Bzl: 2,6-
dichlorobenzyl) with benzotriazol-1-yloxytris(dimethylamino)-
phosphonium hexafluorophosphate21 (BOP) in the presence of
HOBT. Nα-Boc groups were removed by a standard protocol,
20 min treatment with 50% TFA in CH2Cl2. The protected pep-
tide resin was treated with 1 mol dmϪ3 TFMSA–thioanisole in
TFA containing m-cresol at rt for 60 min. There were no dif-
ficulties during the synthesis, and the HPLC profile of the crude
product showed a single major peak at a retention time identi-
cal with that of an authentic sample prepared by the solution
method,22 as shown in Fig. 3. This demonstrates the conformity
of the Chx group to a common Boc-SPPS protocol.
Fig. 3 Analytical HPLC profile of crude Ac-Tyr-Ile-Gly-Ser-Arg-
β-Ala-OH (conditions are given in the Experimental section).
Simulation of SPPS of large peptides
Application of Boc-Thr(Chx)-OH to the conventional peptide
synthesis in solution
The substantial stability of Chx would be advantageous for
improving the purity of the resulting peptides, particulary in
SPPS of large peptides. For exemplification, SPPS of large pep-
tides was simulated by Merrifield’s protocol,16 where three large
peptides from human immunoglobulin Eu17 are used as models.
The yields of three large peptides that lost no protection of Ser
residues were calculated using kChx and kBzl, and the results are
summarized in Table 2. Calculated yields were higher with Chx
than with Bzl, particularly for larger peptides. From these simu-
lations the O-Chx protection is expected to be more favorable
for SPPS of large peptides than is O-Bzl protection.
The segment-condensation strategy, where protected peptide
segments are coupled to afford a larger protected peptide, have
been successfully employed in conventional solution peptide
synthesis.23 A protected peptide segment having the free
carboxy function is required in the segment-condensation
strategy. To prepare such a protected peptide segment, the
carboxy-terminal protecting group of a fully protected peptide
intermediate should be removable without loss of protecting
groups for the α-amino and side-chain functionalities. Since the
J. Chem. Soc., Perkin Trans. 1, 2000, 1949–1954
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Chx group is stable under catalytic hydrogenolytic conditions
as stated above, benzyl-based protecting groups would be
selectively removable from the protected peptides having
Chx side-chain protection.
To demonstrate this, Ac-Asp(OChx)-Thr(Chx)-Thr(Chx)-
Pro-OH, a key intermediate for the synthesis of analogs of an
anti-human immunodeficiency virus type-1 peptide,24 was syn-
thesized using Boc-Thr(Chx)-OH. As summarized in Scheme
2, the fully protected peptide, Boc-Asp(OChx)-Thr(Chx)-
Experimental
Rf -Values in TLC (Kieselgel 60 F254, Merck) refer to CHCl3–
MeOH–AcOH (90:8:2), and spots were detected with UV
light and/or staining with 0.1% ninhydrin in acetone. Optical
rotations were measured with a JASCO DIP-370 polarimeter,
and [α]D-values are in units of 10Ϫ1 deg cm2 gϪ1. HPLC was
conducted with a WATERS 600 system, and A and B in the
mobile-phase system refer to 0.05% aq. TFA and 0.05% TFA in
MeCN, respectively. 1H NMR spectra were recorded on a JEOL
Lambda 400 MHz spectrometer for samples in CDCl3 solution.
J-Values are given in Hz. SiMe4 was used as the internal
standard.
DMF was distilled from ninhydrin before use. Boc-Thr-OH
and Boc-Asp(O-Chx)-OH were purchased from Watanabe
Chemical Industries, Ltd. (Hiroshima, Japan). Che-Br was
purchased from Aldrich Chemical Company (USA), and used
without purification. Fluoren-9-ylmethyl N-succinimidyl carb-
onate (Fmoc-OSu) was purchased from Novabiochem (USA).
Merrifield resin (0.33 mequiv. Cl gϪ1, 1% divinylbenzene-cross-
linked polystyrene, 100–200 mesh) was purchased from Peptide
Institute, Inc. (Osaka, Japan). HOBT used was its hydrate.
Other reagents were of reagent grade and used without
purification.
Boc-Ser(Che)-OHؒCHA**
Scheme 2 Reagents: (i) BOP, Et3N; (ii) 4 mol dmϪ3 HCl in AcOEt;
(iii) acetic anhydride, Et3N; (iv) H2, Pd–charcoal.
To a solution of Boc-Ser-OH (6.2 g, 30 mmol) in DMF (150
cm3) was added NaH (60–72% oil dispersion; 2.4 g, ≈60 mmol)
portionwise under N2 stream at 0 ЊC. After the evolution of
H2 gas had ceased, 3-bromocyclohexene (5.8 g, 36 mmol) was
added to the above solution. The reaction mixture was stirred at
room temperature (rt) overnight. After removal of the solvent
under reduced pressure below 40 ЊC, the residue was dissolved
in water (80 cm3) under cooling with ice, and the solution was
washed with diethyl ether (20 cm3 × 3). The aqueous phase was
acidified to pH 4 with 10% aq. citric acid, and extracted with
CHCl3 (200 cm3). The extract was washed with water, dried over
Na2SO4, and then evaporated to give a yellowish oil (3.9 g,
46%), which was chromatographically pure and could be
used in the next hydrogenation reaction without purification;
δH 5.88–5.85 (1 H, m), 5.74–5.69 (1 H, m), 5.42 (1 H, d, J 6.6),
4.44–4.43 (1 H, m), 4.01–3.95 (1 H, m), 3.92–3.90 (1 H, m),
3.77–3.69 (1 H, m), 2.07–1.91 (2 H, m), 1.85–1.61 (2 H, m),
1.56–1.52 (2 H, m), 1.46 (9 H, s).
Thr(Chx)-Pro-OBzl, was synthesized in a stepwise manner.
Although the amino acid sequence, Asp(OChx)-Thr(Chx)-
Thr(Chx), implies steric hindrance (since the β-branching Thr
residues are consecutive and the Chx group is somewhat bulk-
ier than the Bzl group) satisfactory yields could be acheived
at every coupling step without excess of the carboxylic com-
ponent and coupling reagent. After removal of the amino-
terminal Boc group, the resulting amine was acetylated with
acetic anhydride to give Ac-Asp(OChx)-Thr(Chx)-Thr(Chx)-
Pro-OBzl. The carboxy-terminal Bzl group was then removed
by hydrogenation over Pd–charcoal to give Ac-Asp(OChx)-
Thr(Chx)-Thr(Chx)-Pro-OH in an almost quantitative yield.
This is demonstrative of the usefulness of a Boc/O-Chx/Bzl
protection scheme in the synthesis of protected peptide
segments.
To an ice-cooled solution of a small part of the product (0.80
g) in diethyl ether (50 cm3) was added CHA (0.99 g) to afford
the title salt as a precipitate, which was collected by filtration
and washed with diethyl ether successively; yield 0.98 g; mp
170–175 ЊC (decomp.); [α]D25 ϩ37.2 (c 1.0 in DMF) (Found: C,
61.2; H, 9.40; N, 7.34. C20H36N2O5ؒ0.4H2O requires C, 61.3; H,
9.47; N, 7.15%).
Conclusions
The Chx group could be easily introduced onto the hydroxy
function of Ser and Thr in two steps from Boc-Ser-OH and
Boc-Thr-OH via O-Che derivatives. The Chx group was stable
under various acidic and basic conditions, including deprotec-
tion conditions for Boc, Z, and Fmoc, and thus adequate for
use in combination with any of these Nα-protecting groups. It
could be removed with 1 mol dmϪ3 TFMSA–thioanisole in
TFA in a short period. The stability of the O-Chx group under
common Boc-deprotecting conditions was >20-fold higher than
that of the O-Bzl group. Simulation of SPPS of immuno-
globlin-related large peptides revealed that the Chx group
would be more favorable for use in SPPS of large peptides than
would Bzl. Two peptides containing Ser or Thr were success-
fully synthesized by solid-phase and conventional solution
methods using Chx as side-chain protection. The results
obtained here clearly demonstrate the usefulness of the Chx
group in peptide synthesis. The Chx group is the first sec-alkyl-
based protecting group for the alcoholic hydroxy function. This
new hydroxy-protecting group would be useful not only for pep-
tide synthesis, but also for the synthesis of various compounds
having multiple functional groups, where selective removal of
specific protecting groups is required, because of its unique
stability/removability profile, different from that of any other
hydroxy-protecting group available now.
Boc-Ser(Chx)-OHؒCHA
Boc-Ser(Che)-OH [prepared from Boc-Ser(Che)-OHؒCHA (1.0
g, 2.6 mmol) in the usual manner] was dissolved in a mixture
of MeOH (50 cm3) and water (10 cm3), and hydrogenated in
the presence of PtO2 (0.1 g) for 90 min. After removal of the
solvent, the residue in CHCl3 (5 cm3) was applied to a silica gel
column (Wako Gel C-200, 10 g), which was equilibrated and
eluted with CHCl3. After removal of the solvent from the efflu-
ent (54–378 cm3), the oily residue was dissolved with diethyl
ether. CHA (0.26 g, 2.6 mmol) was added to the solution
to afford the title salt as a precipitate, which was collected by
filtration and washed with diethyl ether; yield 0.67 g (90%); mp
195–198 ЊC (decomp.); [α]D25 ϩ27.2 (c 1.0 in MeOH) (Found: C,
60.8; H, 10.0; N, 7.24. C20H38N2O5ؒ0.5H2O requires C, 60.7; H,
9.94; N, 7.08%); δH (free acid form) 5.40 (1 H, d, J 7.8), 4.43–
** CHA = cyclohexylamine.
1952
J. Chem. Soc., Perkin Trans. 1, 2000, 1949–1954

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4.40 (1 H, m), 3.96–3.93 (1 H, m), 3.66 (1 H, dd, J 4.5 and 9.4),
3.32–3.27 (1 H, m), 1.87–1.83 (2 H, m), 1.73–1.68 (2 H, m),
1.52–1.49 (2 H, m), 1.46 (9 H, s), 1.31–1.20 (4 H, m).
1 mol dm؊3 TFMSA–thioanisole in TFA. Fmoc-Ser(Chx)-OH
(50 µmol) and Fmoc-Ala-OH (50 µmol) were treated with the
title reagent (5.0 cm3) in a similar manner to the above except
for the reaction time (30 min) and temperature (rt).
Boc-Thr(Che)-OHؒCHA
10% Et3N in DMF, 10% aq. NaHCO3, 2 mol dm؊3 aq. NaOH,
and 20% piperidine in DMF. Boc-Ser(Chx)-OH (20 µmol)
and Boc-Ala-OH (20 µmol) were dissolved in the test reagent
(1 cm3), and % survival was determined in a similar manner
to the above.
The title compound was synthesized from Boc-Thr-OH (2.0 g,
8.7 mmol), NaH (60–72% oil dispersion; 0.6 g, ca. 17 mmol),
and 3-bromocyclohexene (1.7 g, 8.7 mmol) in essentially the
same manner as that for Boc-Ser(Che)-OHؒCHA, yield 1.7 g
(62%); mp 167–169 ЊC (decomp.); [α]D25 ϩ3.5 (c 1.0 in DMF)
(Found: C, 54.6; H, 6.49; N, 10.9. C21H38N2O5ؒ1.6H2O requires
C, 54.4; H, 6.88; N, 11.3%); δH (free acid form) 5.89–5.86 (1 H,
m), 5.73–5.65 (1 H, m), 5.38 (1 H, d, J 6.8), 4.35–4.32 (1 H, m),
4.22–4.20 (1 H, m), 4.03–3.97 (1 H, m), 2.06–1.55 (6 H, m), 1.46
(9 H, s), 1.22 (3 H, d, J 6.4).
H2/Pd. Boc-Ser(Chx)-OH (20 µmol) and Boc-Ala-OH (20
µmol) were dissolved in MeOH–water (4:1 v/v; 1 cm3). To the
solution was added a small amount of Pd–charcoal. The sus-
pension was stirred under H2 atomosphere at rt. Parts of the
solution were collected at 2 and 8 h, passed through cotton
filters individually, and subjected to HPLC. % Survival was
determined in a similar manner to the above.
Boc-Thr(Chx)-OH
The title compound was obtained from Boc-Thr(Che)-OHؒ
CHA (1.7 g, 4.3 mmol) in essentially the same manner as that
for Boc-Ser(Chx)-OH as a colorless amorphous powder (1.3 g,
79%); [α]D25 ϩ2.5 (c 1.0 in DMF) (Found: C, 56.1; H, 6.36; N,
11.0. C15H27NO5ؒ2H2O requires C, 55.9; H, 6.68; N, 11.4%);
δH 5.33 (1 H, d, J 7.8), 4.29 (1 H, dd, J 3.1 and 7.9), 4.17–4.14
(1 H, m), 3.42–3.38 (1 H, m), 1.81–1.78 (2 H, m), 1.68–1.65
(2 H, m), 1.49–1.46 (2 H, m), 1.43 (9 H, s), 1.30–1.20 (4 H, m),
1.15 (3 H, d, J 6.4).
Determination of kChx
Fmoc-Ser(Chx)-OH (50 µmol) and Fmoc-Ala-OH (50 µmol)
were dissolved in 50% TFA in CH2Cl2 (5.0 cm3), and the solu-
tion was stored at 25 ЊC in a glass tube with a tight cap and
Teflon sealing. At 0, 2, 6, 24, 48, 96, and 192 h after mixing,
parts of the solution were subjected to HPLC [column,
WATERS µBondasphere 5C18 (3.9 × 150 mm); solvent, A:B
80:20 to 20:80 in 60 min (1.0 cm3 minϪ1)], and peak areas of
Fmoc-Ser(Chx)-OH (tR 39.23 min) and Fmoc-Ala-OH (tR
25.45 min) were determined. The X0/Xt-value was corrected
using the internal standard; X0/Xt = (S0ؒAt)/(StؒA0). The best-fit
line through data points plotted on a grid of ln (X0/Xt) vs. t
is shown in Fig. 2. The rate constant, kChx, was determined as
0.46 × 10Ϫ8 sϪ1 from its slope.
Fmoc-Ser(Chx)-OH
To a solution of Fmoc-OSu (203 mg, 0.60 mmol) in 1,2-
dimethoxyethane (50 cm3) was added a solution of H-Ser(Chx)-
OHؒHCl [prepared from Boc-Ser(Chx)-OHؒCHA (259 mg,
0.67 mmol) and 4 mol dmϪ3 HCl in AcOEt (2.0 cm3) in the
usual manner] in 5% aq. NaHCO3 (20 cm3) portionwise. The
resulting mixture was stirred at rt overnight. After removal of
the insoluble substance, the solution was concentrated to a
third of the initial volume with a rotary evaporator. The con-
densed solution was acidified with 6 mol dmϪ3 aq. HCl under
cooling with ice, and then extracted with AcOEt. The extract
was washed with water, dried over Na2SO4, and evaporated to
dryness. n-Hexane was added to the oily residue to afford
crystals of the title acid, which were collected by flitration
and recrystallized from CHCl3–n-hexane, yield 220 mg
(89.5%), mp 155–157 ЊC; [α]D25 –27.0 (c 0.5, DMF) (Found: C,
70.55; H, 6.81; N, 3.56. C24H27NO5 requires C, 70.4; H, 6.65;
N, 3.42%).
Simulation of SPPS of large peptides related to human
immunoglobulin Eu
The yields of three large peptides that maintain complete
protection of Ser residues were calculated as described in a
previous report.16
Boc-ꢀAla-O-Merrifield resin
Boc-βAla-OH (0.31 g, 1.65 mmol) was converted to its Cs salt
in the usual manner.25 Boc-Ala-OCs was added to a suspension
of Merrifield resin (0.33 mequiv. gϪ1; 5 g) in DMF (50 cm3). The
mixture was stirred gently at rt for 4 h. The resin was collected
by filtration, washed successively with DMF, CH2Cl2, MeOH,
and n-hexane, and dried over CaCl2 in vacuo, yield 5.24 g [0.30
mmol βAla gϪ1 (determined by the weight increase)].
Stability and removability of the Chx group
Ac-Tyr-Ile-Gly-Ser-Arg-ꢀAla-OH
TFA and 4 mol dm؊3 HCl in AcOEt. Fmoc-Ser(Chx)-OH (20
µmol) and Fmoc-Ala-OH (20 µmol) were dissolved in the test
reagent (1.0 cm3). Parts of the solution were collected at times
given in Table 1, and subjected to HPLC. The percentage
survival of the Chx group was determined as follows: %
survival = 100 × (St × A0/At)/S0, where S0 is the peak area of
Fmoc-Ser(Chx)-OH on analysis of the initial solution; St is
the peak area of Fmoc-Ser(Chx)-OH on analysis of the test
solution stored for t h; A0 is the peak area of Fmoc-Ala-OH
on analysis of the initial solution; and At is the peak area
of Fmoc-Ala-OH on analysis of the test solution stored for
t h.
Starting from Boc-βAla-O-Merrifield resin (500 mg, 0.15
mmol), SPPS was carried out by the following protocol: 1)
CH2Cl2 (5 cm3), 1 min × 5; 2) 50% TFA in CH2Cl2 (5 cm3), 2
min × 1, 20 min × 1; 3) CH2Cl2 (5 cm3), 1 min × 5, then DMF
(5 cm3), 1 min × 5; 4) Boc-amino acid (0.45 mmol), BOP (0.45
mmol), HOBT (0.45 mmol), EtNPri2 (0.45 mmol), DMF (5
cm3), 30 min; 5) DMF (5 cm3), 1 min × 5; repeat from step 1).
After removal of the terminal Boc group, acetic anhydride (0.45
mmol) and EtNPri2 (0.45 mmol) were added to the resin in
DMF (5 cm3), and the mixture was agitated for 60 min. The
resulting peptide resin was washed as follows: DMF (5 cm3), 1
min × 5; CH2Cl2 (5 cm3), 1 min × 5; MeOH, 1 min × 5; and
then n-hexane (5 cm3), 1 min × 5, and dried over CaCl2
in vacuo, yield 587 mg.
To an ice-cooled suspension of the peptide resin obtained in
TFA (3.8 cm3) containing thioanisole (0.59 cm3, 5.0 mmol) and
m-cresol (0.17 cm3) were added TFMSA (0.44 cm3, 5.0 mmol).
The mixture was gently stirred at ice-bath temperature for 30
min, and then at rt for 60 min. Diethyl ether (50 cm3) and
water (20 cm3) were added to the reaction mixture. The water
1 mol dm؊3 TMSBr–thioanisole in TFA. Fmoc-Ser(Chx)-OH
(50 µmol) and Fmoc-Ala-OH (50 µmol) were dissolved in TFA
(5.0 cm3). A part of the solution (0.6 cm3) was stored as a
standard. To the remaining part of the solution cooled with an
ice-bath were added thioanisole (290 mm3) and TMSBr (310
mm3). The mixture was stirred at ice-bath temperature. After 30
and 120 min, parts of the solution were subjected to HPLC. %
Survival of the Chx group was determined as described above.
J. Chem. Soc., Perkin Trans. 1, 2000, 1949–1954
1953

View Article Online
layer was washed with diethyl ether (50 cm3 × 5), neutralized
with NaHCO3, and applied to a Sephadex G-10 column
(2.8 × 48 cm), which was equilibrated and eluted with 3% aq.
AcOH. Individual fractions (11 cm3) were collected, and the
desired fractions (#10–15) were combined and lyophilized to
give a white fluffy powder (64 mg, 60% from the starting resin);
tR = 15.38 min [90.0%, column YMC-R-ODS (4.6 × 250 mm),
A:B 95:5 to 20:80 in 45 min (1.0 cm3 minϪ1), the major peak
was confirmed to co-elute with an authentic sample22 prepared
by the conventional solution method].
and evaporated to dryness. Diethyl ether was added to the resi-
due to afford crystals of the title product, which were collected
by filtration; yield 1.8 g (87%); mp 133–137 ЊC; [α]D25 Ϫ19.9 (c 1.0,
DMF) (Found: C, 59.7; H, 8.52; N, 6.48. C43H66N4O10ؒ3.8H2O
requires C, 59.5; H, 8.55; N, 6.46%).
Ac-Asp(OChx)-Thr(Chx)-Thr(Chx)-Pro-OH
Ac-Asp(OChx)-Thr(Chx)-Thr(Chx)-Pro-OBzl (1.2 g, 1.5
mmol) was dissolved in MeOH–water (10:1 v/v; 110 cm3), and
hydrogenated in the presence of Pd–charcoal (5%; 1.0 g) at rt
for 4 h. After removal of the catalyst and solvent, the residue
was extracted with AcOEt (100 cm3), and the extract was dried
over Na2SO4, and evaporated to dryness. Diethyl ether was
added to the residue to afford crystals of the title acid, which
were collected by filtration; yield 1.0 g (93%); mp 161–165 ЊC;
[α]D25 Ϫ19.8 (c 1.0, DMF) (Found: C, 48.7; H, 9.02; N, 6.70.
C34H60N4O11ؒ7.6H2O requires C, 48.7; H, 9.05; N, 6.69%).
H-Thr(Chx)-Pro-OBzlؒHCl
To an ice-cooled solution of H-Pro-OBzlؒHCl (3.4 g, 14 mmol)
and Boc-Thr(Chx)-OH (3.7 g, 14 mmol) in DMF (200 cm3)
containing Et3N (2.0 cm3, 14 mmol) were added BOP (6.2 g, 14
mmol) and Et3N (4.0 cm3, 28 mmol). The mixture was stirred at
rt overnight. After removal of the solvent, the residue was
extracted with AcOEt (200 cm3). The extract was washed suc-
cessively with 5% aq. NaHCO3, 10% aq. citric acid, and water,
dried over Na2SO4, and evaporated to dryness. The oily residue
was dissolved in 4 mol dmϪ3 HCl in AcOEt (14 cm3). The solu-
tion was stirred at ice-bath temperature for 1 h and then at rt for
1 h, and then evaporated. Diethyl ether was added to the resi-
due to afford crystals of the title salt, which were collected by
filtration; yield 4.0 g (70%); mp 174–176 ЊC; [α]D25 Ϫ57.7 (c 1.0,
DMF) (Found: C, 51.6; H, 8.38; N, 5.40. C22H33ClN2O4ؒ
4.9H2O requires C, 51.5; H, 8.38; N, 5.40%).
References
1 Preliminary communication, Y. Nishiyama and K. Kurita, Tetra-
hedron Lett., 1999, 40, 927.
2 Practical guidelines for side-chain protections in solid-phase
peptide synthesis: E. Atherton and R. C. Sheppard, Solid Phase
Peptide Synthesis–A Practical Approach, IRL Press, Oxford, 1989;
G. Barany and R. B. Merrifield, in The Peptides: Analysis, Synthesis,
Biology. Vol. 2. Special Methods in Peptide Synthesis Part A,
ed. E. Gross and J. Meienhofer, Academic Press, New York, 1979,
pp. 1–284.
3 A. Furka, in Combinatorial Peptide and Nonpeptide Libraries,
ed. G. Jung, VCH Verlagsgesellschaft mbH, Weinheim, 1996,
pp. 111–137.
4 For example, B. W. Bycroft, W. C. Chan, S. R. Chhabra and
N. D. Hone, J. Chem. Soc., Chem. Commun., 1993, 778.
5 For example, S. Futaki, T. Takike, T. Akita and K. Kitagawa,
J. Chem. Soc., Chem. Commun., 1990, 523.
6 J. M. Stewart, in The Peptides: Analysis, Synthesis, Biology. Vol. 3.
Protection of Functional Groups in Peptide Synthesis, ed. E. Gross
and J. Meienhofer, Academic Press, New York, 1981, pp. 169–201.
7 H. Yajima, N. Fujii, H. Ogawa and H. Kawatani, J. Chem. Soc.,
Chem. Commun., 1974, 107.
8 D. Yamashiro, J. Org. Chem., 1977, 42, 523.
9 V. S. Chauhan, S. T. Ratcliffe and G. T. Young, Int. J. Pept. Protein
Res., 1980, 15, 96.
10 J. P. Tam, T.-W. Wong, M. W. Riemen, F.-S. Tjoeng and R. B.
Merrifield, Tetrahedron Lett., 1979, 4033.
11 J. P. Tam, M. W. Riemen and R. B. Merrifield, Pept. Res., 1988, 1, 6.
12 M. Engelhard and R. B. Merrifield, J. Am. Chem. Soc., 1978, 100,
3559.
H-Thr(Chx)-Thr(Chx)-Pro-OBzlؒHCl
To an ice-cooled solution of H-Thr(Chx)-Pro-OBzlؒHCl (2.9 g,
7.1 mmol) and Boc-Thr(Chx)-OH (1.8 g, 7.1 mmol) in DMF
(200 cm3) containing Et3N (1.0 cm3, 7.1 mmol) were added BOP
(3.1 g, 7.1 mmol) and Et3N (2.0 cm3, 14 mmol). The mixture
was stirred at rt overnight. After removal of the solvent, the
residue was extracted with AcOEt (200 cm3). The extract was
washed successively with 5% aq. NaHCO3, 10% aq. citric acid,
and water, dried over Na2SO4, and evaporated to dryness. The
oily residue was dissolved in 4 mol dmϪ3 HCl in AcOEt (8.9
cm3). The solution was stirred at ice-bath temperature for 1 h
and then at rt for 1 h, and then evaporated. Diethyl ether was
added to the residue to afford crystals of the title salt, which
were collected by filtration; yield 3.9 g (90%); mp 115–119 ЊC;
[α]D25 Ϫ63.2 (c 1, DMF) (Found: C, 56.8; H, 8.55; N, 6.08.
C32H50ClN3O6ؒ4H2O requires C, 56.5; H, 8.59; N, 6.18%).
13 Y. Nishiuchi, H. Nishio and S. Sakakibara, Tetrahedron Lett., 1996,
37, 7529.
14 H. Sugano and M. Miyoshi, J. Org. Chem., 1976, 41, 2352.
15 N. Fujii, A. Otaka, N. Sugiyama, M. Hatano and H. Yajima, Chem.
Pharm. Bull., 1987, 35, 3880.
16 B. W. Erickson and R. B. Merrifield, J. Am. Chem. Soc., 1973, 95,
3750.
17 G. M. Edelman, B. A. Cunningham, W. E. Gall, P. D. Gottlieb,
U. Rutishauser and M. J. Waxdal, Proc. Natl. Acad. Sci. USA, 1969,
63, 78.
18 Y. Iwamoto, F. A. Robey, J. Graf, M. Sasaki, H. K. Kleinman,
Y. Yamada and G. R. Martin, Science, 1987, 238, 1132.
19 R. B. Merrifield, Biochemistry, 1964, 3, 1385.
20 C. H. Li, J. Meienhofer, E. Schnabel, D. Chung, T.-B. Lo and
J. Ramachandran, J. Am. Chem. Soc., 1961, 83, 4449.
21 B. Castro, J. R. Dormoy, G. Evin and C. Selve, Tetrahedron Lett.,
1975, 1219.
22 Y. Nishiyama, T. Yoshikawa, N. Ohara, K. Kurita, K. Hojo,
H. Kamada, Y. Tsutsumi, T. Mayumi and K. Kawasaki, J. Chem.
Soc., Perkin Trans. 1, 2000, 1161.
23 For recent examples, see Y. Nishiuchi, T. Inui, H. Nishio, J. Bodi,
T. Kimura, F. I. Tsuji and S. Sakakibara, Proc. Natl. Acad. Sci.
USA, 1998, 95, 13549; H. Nishio, T. Inui, Y. Nishiuchi, C. L. De
Medeiros, E. G. Rowan, A. L. Harvey, E. Katoh, T. Yamazaki,
T. Kimura and S. Sakakibara, J. Pept. Res., 1998, 51, 355.
24 Y. Nishiyama, T. Murakami, K. Kurita and N. Yamamoto, Bioorg.
Med. Chem. Lett., 1999, 9, 1357.
Boc-Asp(OChx)-Thr(Chx)-Thr(Chx)-Pro-OBzl
To an ice-cooled solution of H-Thr(Chx)-Thr(Chx)-Pro-
OBzlؒHCl (2.5 g, 4.1 mmol) and Boc-Asp(OChx)-OH (1.3 g,
4.1 mmol) in DMF (200 cm3) containing Et3N (0.60 cm3, 4.1
mmol) were added BOP (1.8 g, 4.1 mmol) and Et3N (1.2 cm3,
8.2 mmol). The mixture was stirred at rt overnight. After
removal of the solvent, the residue was extracted with AcOEt
(200 cm3). The extract was washed successively with 5% aq.
NaHCO3, 10% aq. citric acid, and water, dried over Na2SO4,
and evaporated to give the title compound as an amorphous
powder (3.2 g, 90%); [α]D25 Ϫ42.1 (c 1.0, DMF) (Found: C, 60.3;
H, 8.50; N, 6.06. C47H72N4O11ؒ3.5H2O requires C, 60.6; H, 8.54;
N, 6.01%).
Ac-Asp(OChx)-Thr(Chx)-Thr(Chx)-Pro-OBzl
To an ice-cooled solution of H-Asp(OChx)-Thr(Chx)-
Thr(Chx)-Pro-OBzlؒHCl [prepared from Boc-Asp(OChx)-
Thr(Chx)-Thr(Chx)-Pro-OBzl (2.0 g, 2.5 mmol) and 4 mol
dmϪ3 HCl in AcOEt (4.6 cm3) in the usual manner] in DMF
(100 cm3) containing Et3N (0.40 cm3, 2.5 mmol) was added
acetic anhydride (0.3 cm3, 2.7 mmol). The mixture was stirred
at rt for 2 h. After removal of the solvent, the residue was
extracted with AcOEt (200 cm3). The extract was washed suc-
cessively with 10% aq. citric acid and water, dried over Na2SO4,
25 S.-S. Wang, B. F. Gisin, D. P. Winter, R. Makofske, I. D. Kulesha,
C. Tzougraki and J. Meienhofer, J. Org. Chem., 1977, 42, 1286.
1954
J. Chem. Soc., Perkin Trans. 1, 2000, 1949–1954