1-Ethyl 2-halopyridinium salts, highly efficient coupling reagents for hindered peptide synthesis both in solution and the solid-phase

Article abstract of DOI:10.1016/S0040-4020(00)00657-8

1-Ethyl-2-halopyridinium salts, BEP, FEP, BEPH and FEPH, were synthesized and proved to be very effective for the synthesis of hindered peptides containing N-methylated or C(α),C(α)-dialkylated amino acid residues. HPLC monitoring of model reactions indicated that these pyridinium salts demonstrated higher reactivities, lower racemization than the commonly used halogenated uronium and phosphonium salts. The efficiency of these pyridinium type coupling reagents was further proved by the synthesis of a series of hindered oligopeptides and active esters with good yields and convenient workup. The 8-11 tetrapeptide fragment of Cyclosporin A (CsA) and the pentapeptide moiety of Dolastatin 15 were also successfully synthesized using these pyridinium salts. The efficiency of these pyridinium type coupling reagents for SPPS was also demonstrated by the solid-phase synthesis of the extremely hindered 8-11 peptide segment of CsA and the linear undecapeptide of CsO. The mechanism of the pyridinium salt mediated coupling reactions was also studied by ¹H NMR, IR and HPLC. It was proposed that the major reactive intermediates were the corresponding acyl halide and acyloxypyridinium salts of the N-protected amino acid or peptide. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00657-8

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 8119±8131
1-Ethyl 2-Halopyridinium Salts, Highly Ef®cient Coupling
Reagents for Hindered Peptide Synthesis both in Solution
and the Solid-Phase
*
Peng Li and Jie-Cheng Xu
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China
Received 18 May 2000; revised 5 July 2000; accepted 20 July 2000
AbstractÐ1-Ethyl-2-halopyridinium salts, BEP, FEP, BEPH and FEPH, were synthesized and proved to be very effective for the synthesis
of hindered peptides containing N-methylated or Ca,Ca-dialkylated amino acid residues. HPLC monitoring of model reactions indicated that
these pyridinium salts demonstrated higher reactivities, lower racemization than the commonly used halogenated uronium and phosphonium
salts. The ef®ciency of these pyridinium type coupling reagents was further proved by the synthesis of a series of hindered oligopeptides and
active esters with good yields and convenient workup. The 8±11 tetrapeptide fragment of Cyclosporin A (CsA) and the pentapeptide moiety
of Dolastatin 15 were also successfully synthesized using these pyridinium salts. The ef®ciency of these pyridinium type coupling reagents
for SPPS was also demonstrated by the solid-phase synthesis of the extremely hindered 8±11 peptide segment of CsA and the linear
1
undecapeptide of CsO. The mechanism of the pyridinium salt mediated coupling reactions was also studied by H NMR, IR and HPLC.
It was proposed that the major reactive intermediates were the corresponding acyl halide and acyloxypyridinium salts of the N-protected
amino acid or peptide. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
unhindered amide bonds, while the chain assembly of
sterically hindered peptides containing N-methyl or Ca,Ca-
dialkyl amino acid residues is inef®cient using the above
reagents except the HOAt-derived onium salts.14 However,
these HOAt-based reagents are expensive and unsuitable for
large scale peptide synthesis, and may react with amino
components to form the corresponding guanidinium deriva-
tives, especially in peptide segment condensation and cycli-
zation.15 As an alternative approach, halogenated coupling
reagents, such as PyBroP,16 BroP,17 CIP,18 TFFH,19
BTFFH,20 PyCIU,4b CDTP,21 CMMM, BOP-Cl,22 and
BEMT23 shown in Fig. 2, were also found to be ef®cient
In the past decades, many new peptide coupling reagents
have been designed, synthesized and commonly used in
peptide synthesis. Among these reagents, HOBt- and
HOAt-based uronium, phosphonium and immonium salts,
such as BOP,1 HBTU,2 PyBOP,3 HBPyU,4 HATU,5
HAPyU,6 AOP,7 PyAOP,8 BOMI,9 BDMP,10 BDMP,11 and
AOMP have been proven to be very ef®cient (Fig. 1). The
predominance of carbodiimide12 and active ester tech-
niques13 have been gradually replaced with onium salts.
These reagents can ef®ciently promote the formation of
Keywords: 1-ethyl-2-halopyridinium; cyclosporin A; dolastatin 15.
* Corresponding author. Tel.: 186-21-6416-3300-1235; fax: 186-21-6416-6128; e-mail: xjcheng@pub.sioc.ac.cn
Abbreviations: Aib, a-aminoisobutyric acid; AOMP, 5-(1H-7-azabenzotriazol-l-yloxy)-3,4-dihydro-l-methyl 2H-pyrrolium hexachloroantimonate; AOP, (1H-
7-azabenzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexa¯uoroposphate; BDMP, 5-(1H-Benzotriazol-1-yl)-3,4-dihydro-1-methyl 2H-pyrrolium
hexachloroantimonate: N-oxide; BEMT, 2-bromo-3-ethyl-4-methyl thiazolium tetra¯uoroborate; BEP, 2-bromo-1-ethyl pyridinium tetra¯uoroborate;
BEPH, 2-bromo-1-ethyl pyridinium hexachloroantimonate; BOMI, N-(1H-benzotriazol-1-ylmethylene)-N-methylmethanaminium hexachloroantimonate N-
oxide; BOP, (1H-benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexa¯uorophosphate; BOP-Cl, N,N0-bis(2-oxo-3-oxazolidinyl)phosphinic chloride;
BPMP, 1-(1H-benzotriazol-1-yloxy)phenylmethylene pyrrolidinium hexachloroantimonate; BTFFH, 1,1,3,3-bis(tetramethylene) ¯orouronium hexa¯uoro-
phosphate; CMMM, chloro(4-morphoino)methylene morpholinium hexa¯uorophosphate; DCC, dicyclohexlcarbodiimide; DIEA, N,N(-diisopropylenthyl-
amine; DMAP, 4-dimethylaminopyridine; FEP, 2-¯uoro-1-ethyl pyridinium tetra¯uoroborate; FEPH, 2-¯uoro-1-ethyl pyridinium hexachloroantimonate;
HAPyU, 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)pyrrolidinium hexa¯uorophosphate N-oxide; HATU, N-[(dimethylamino)-1H-
1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)-N-methylmethanaminium hexa¯uoro-phosphate N-oxide; HBPipU, O-(1H-benzotriazol-l-yl)-N,N,N0,N0-
bis(pentamethylene)uronium hexa¯uoro-phosphate; HBPyU O-(1H-benzotriazol-1-yl)-N,N,N0,N0-bis(tetramethylene)urinium hexa¯uorophosphate;; HBTU,
N-[(1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexa¯uorophosphate N-oxide; HOAt, 1-hydroxy-7-azabenzotriazole;
HOBt, 1-hydroxybenzotriazole; PE, petroleum; PyAOP, (1H-7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexa¯uorophosphate; PyBOP, (1H-
benzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexa¯uorophosphate; PyBrop, bromotrpyrrolidinophosphonium hexa¯uorophosphate; PyCloP, chlorotri-
pyrrolidinophosphonium hexa¯uorophosphate; PyClU, 1,1,3,3-bis(tetramethylene) chlorouronium hexa¯uorophosphate; TFFH, tetramethyl¯uoromamidi-
nium hexa¯uorophosphate. Nomenclature and symbols of amino acids and peptides generally follow the recommendations of the IUPAC-IUB Joint
Commission of Biochemical Nomenclature in: Pure Appl. Chem. 1984, 56, 595±624.
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00657-8

8120
P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131

P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
8121
Figure 2. The structures of the halogenated peptide coupling reagents.
Scheme 1. Synthesis of 1-ethyl-2-halopyridinium salts.
for the synthesis of hindered peptides, especially for the
scale-up of the preparation of peptides since they are inex-
pensive. Unfortunately, these halogenated reagents, except
BOP-Cl and BEMT, usually result in high racemization
during coupling, especially for segment condensation.
Therefore, it is necessary and signi®cant to develop more
ef®cient and inexpensive coupling reagents to meet the
needs of the synthesis of increasingly challenging peptides
and peptidomimetics, as well as the establishment of peptide
libraries.
ef®ciency.23 Herein, we attempted to replace one nitrogen
with a carbon atom. Considering meantime considered the
stability of generated molecules, it was obvious that the
a-halopyridinium salts were the most suitable candidates.
Mukaiyama et al., have studied 2-halo and 2-bromo-
pyridinium iodides, and used them to synthesize esters,24
lactones25 and carboxamides.26 However, the condensation
reactions had to be carried out under the condition of re¯ux-
ing in methylene chloride due to the poor solubility of
these pyridinium iodides in conventional solvents. Such
Table 1. Comparison of racemization and reactivity of 2-halopyridinium
salts with other coupling reagents (model reaction: Z-Gly-Phe-OH1Val-
OCH3´HCl!Z-Gly-Phe-Val-OCH3; reaction conditions: solvent CH2Cl2
(0.1 M); temp. 2108C; base DIEA)
Results and Discussion
Our molecular design was based upon the general structures
of the halouronium salts since the ef®ciency of the uronium
type coupling reagents seemed slightly better than the corre-
sponding phosphonium analogues.7 Analyzing the struc-
tures of these halouronium salts, we can see that the
carbocation of each uronium salt is well stabilized by the
two conjoint nitrogens and the lone electron pairs of
the nitrogen atoms are delocalized within the N±C±N
bonds, which causes the carbocation to share a relatively
high electron density; consequently the uronium salt
demonstrates relatively low reactivity in the nucleophilic
reaction involved in peptide synthesis. Therefore, we tried
to replace one nitrogen with other atoms without lone elec-
tron pairs or more electronegative atoms with lone electron
pairs to enhance the reactivity of the reaction-mediated
carbocations in uronium molecules. In previous studies,
we replaced one nitrogen with sulfur to develop a
2-halothiazolium type reagent BEMT, and proved its high
Coupling reagent Yield % (tˆ2 min)
t
1/2 (min) d-isomer content (%)
PyBroP
BOP-Cl
PyClU
BTFFH
CMMM
BEMT
BEMTc
FEP
6.1
5.3
5.6
9.4
79
t90a
22.3
4.1
.120 33.2
49
.120 31.4
25.9
2.3
b
45.9
84.3
75.6
67.7
50.8
52.3
86.7
75.9
±
2.7
1.3
2.3
2.1
4.6
4.6
1.5
1.4
,2b
,2b
,2b
,2b
,2b
,2b
,2b
FEPH
BEP
BEPH
FEPc
BEPc
a The t1/2 value of BOP±Cl cannot be evaluated accurately due to its poor
solubility in CH2Cl2.
b The coupling reactions were accomplished within 2 min.
c HOAt (1 equiv.) was added as an additive.

8122
P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
Figure 3. Comparison of reactivity of FEP with other coupling reagents (the reaction model and conditions were the same as in Table 1).
conditions were too rigorous and unsuitable for the syn-
thesis of peptides, especially bio-active peptides. To
improve the solubility of these pyridinium compounds, the
tetra¯uoroborate and hexachloroantimonate counterions
were adopted. To further enhance the reactivity of these
compounds, 2-¯uoropyridinium salts were also selected as
peptide coupling reagent candidates since the rate of
hydrolysis of the 2-¯uoropyridiniums were much faster
than those of 2-bromo or 2-chloro substituted analogues.27
Based upon the above considerations, we designed 2-halo-
pyridinium salts BEP, FEP, BEPH, and FEPH for evaluation
of their ef®ciency in peptide synthesis.
To evaluate the ef®ciency of these pyridinium salts in
peptide synthesis, the model reaction of [211] segment
condensation,
Z-Gly-Phe-OH1Val-OCH3´HCl!Z-Gly-
Phe-Val-OCH3, was selected and monitored by HPLC.
The reactions were carried out in dilute solutions
(0.1 mol/L) to ensure all substrates and products were well
dissolved in the solution. Table 1 and Fig. 3 showed that
these 2-halopyridinium salts were much more reactive than
widely used halogenated reagents, such as PyBroP, PyClU,
BTFFH and BOP-Cl. The coupling was accomplished
within 2 min under the tested reaction conditions as
shown in Fig. 3. The coupling yields were further improved
if base was added slowly or dropwise to avoid the 2-halo-
pyridinium salts reacting too violently. It is obvious that the
racemization of product using 2-halopyridinium salts was
much lower than those of other halogenated coupling
reagents as shown in Table 1 except the reagent BOP-Cl
and BEMT. If HOAt was added as an additive, the
Compared to other halogenated coupling reagents, the
2-halopyridinium salts can be more readily synthesized by
N-alkylation of 2-halopyridine using triethyloxonium tetra-
¯uoroborate or hexachloroantimonate with nearly quantita-
tive yield as colorless shelf-stable crystals (Scheme 1).
Table 2. Preparation of oligopeptides using 2-halopyridinium type coupling reagents (the CO±NH bond formed in the peptide is indicated by p, all products
were con®rmed by H NMR, EIMS and other characterization)
1
Peptide
Reagent
Yield (%)a
Mp (8C)
38±39
Oil
124±126
Oil
Oil
Oil
108±109
Oil
131±132
Oil
[a]D (conc., solv., temp.)
Fmoc-Nvap-Sar-OBzl
Z-MeValp-MeVal-OMe
Fmoc-Leup-Ala-OBzl
Fmoc-Valp-MeVal-OCH3
Fmoc-MeLeup-MeVal-OCH3
Boc-Prop-Pro-OBzl
BEP
BEP
FEP
BEP
FEP
BEP
BEP
BEP
FEP
BEP
98
95
91
88
95
90
96
91
97
48
29.3 (0.3, CHCl3, 238C)
2206 (1, MeOH, 198C)
247.9 (1, MeOH, 208C)
286.0 (0.3, CHCl3, 238C)
2135.6 (1, MeOH, 208C)
2109.4 (1, CHCl3, 238C)
±
2122 (0.2, CHCl3, 228C)
256.3 (1, MeOH, 208C)
2114.8 (1, MeOH, 208C)
Z-Aibp-Aib-OCH3
Fmoc-MeLeup-MeVal-OBzl
Z-Valp-Leu-Ala-OBut
Fmoc-MeLeup-MeLeu-
MeVal-OBzl
Z-Leup-Val-Leu-Ala-OBut
Fmoc-d-Alap-MeLeu-
MeLeu-MeVal-OBzl
Boc-Val-Val-MeValp-Pro-
Pro-OBzl
BEP
BEP
92
94
144±146
36±37
262.1 (1, MeOH, 208C)
2145.4 (0.5, CHCl3, 258C)
BEP
88
89±90
2181 (0.3, CHCl3, 238C)
Boc-Valp-MeVal-OMe
Boc-Prop-Pro-OBzl
FEP
FEP
FEP
FEP
92
94
89
95
Oil
Oil
Glassy solid
108±110
2136.0 (1, MeOH, 208C)
2127.4 (1, MeOH, 218C)
238.2 (1, MeOH, 208C)
±
Z-Leup-Ala-OBut
Z-Aibp-Aib-OCH3
a Isolated yield based upon N a-protected amino acid or peptide.

P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
Table 3. Synthesis of active esters using 2-halopyridinium salts
8123
Substrate
Reagent
Producta
Yield (%)b
Mp (8C)
Carboxylic acid
Alcohol
Boc-Aib-OH
Boc-Aib-OH
Fmoc-Sar-OH
Fmoc-Sar-OH
Fmoc-Sar-OH
CBZ-Ala-OH
HOBt
KOPfp
HOSu
HOOBt
HOPcp
But-OH
FEP
FEP
FEP
BEP
BEPH
FEP
Boc-Aib-OBt
82
76
81
77
85
64
Oil
Boc-Aib-OPfp
Fmoc-Sar-OSu
Fmoc-Sar-OOBt
Fmoc-Sar-OPcp
CBZ-Ala-OBut
105±106
Glassy solid
Glassy solid
113±114
Oil
a All products were con®rmed by 1H NMR, EIMS and other characterization.
b Isolated yield based on carboxylic acid component.
racemization could be further suppressed due to its anchi-
meric assistance effect.28
oligopeptides were prepared as shown in Table 2. In a
typical experimental procedure, DIEA (3.2 equiv.) was
added to a cooled mixture (2108C) of Na-protected
amino acid (1 equiv.), amino acid ester hydrochloride
(1.1 equiv.), and 2-halopyridinium salt (1.1 equiv.) in
In order to evaluate the ef®ciency of these pyridinium type
coupling reagents for hindered peptide synthesis, a series of
Scheme 2. Synthesis of the 8±11 fragment of Cyclosporin A using reagent BEP.

8124
P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
Scheme 3. Synthesis of pentapeptide moiety of Dolastatin 15.
CH2Cl2 (3±5 mL/mmol), stirred for 1 min cold and for 1 h
at room temperature. The reaction time should be moder-
ately prolonged for the coupling between N-methyl amino
acids monitoring by TLC. The products can be easily puri-
®ed by washing with 5% sodium bicarbonate, brine, 0.5 M
citric acid and water successively to remove the byproducts
N-ethyl-2-pyridine and other impurities.
salts in hindered peptide couplings, two extensively
N-alkylated peptide segments were synthesized successfully
using these reagents. During the synthesis of the 8±11 tetra-
peptide fragment of Cyclosporin A, the C-terminus was
intentionally protected as a benzyl ester to investigate the
capability of these pyridinium type reagents, such as BEP,
in preventing the spontaneous formation of diketopipera-
zine. Under this unfavorable circumstance, the tripeptide
Fmoc-MeLeu-MeLeu-MeVal-OBzl 6 was still obtained in
48% yield by sequential deprotection and coupling of Fmoc-
These pyridinium type coupling reagents can also be used to
prepare amides and esters, especially active esters and
hindered esters, such as benzotriazolyl ester, penta¯uoro-
phenyl ester, succinimidyl ester, 2,3-dihydro-4-oxobenzo-
triazinyl ester and tert-butyl ester (Table 3). These active
esters are commonly used in the synthesis of lactones and
lactams.
MeLeu-MeVal-OBzl
5 with Fmoc-MeLeu-OH using
reagent BEP (Scheme 2). According to the report by
Wenger during the synthesis of Cyclosporin A using modi-
®ed mixing pivalic anhydride method, the same desired
tripeptide was not obtained at all due to the spontaneous
formation of diketopiperazine.29 This encouraging result
indicated that BEP was such a powerful coupling
To further demonstrate the ef®ciency of 2-halopyridinium

P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
8125
Scheme 4. Synthesis of the 8±11 segment of Cyclosporin A in the solid-phase using BEP.
reagent that could ef®ciently promote the formation of
hindered peptide bonds, even preferentially to spontaneous
diketopiperazine formation.
Using these a-halopyridinium type reagents, we also
synthesized the hindered pentapeptide moiety of Dolastatin
15, which is a pseudopeptide bearing promising antineo-
plastic activity.30 The [312] segment condensation strategy
was adopted and the Boc group was used for the Na-protec-
tion, thus a 58% overall yield of the protected pentapeptide
14 was obtained via seven coupling steps. After removal of
the Boc protecting group with tri¯uoroacetic acid, the ®nal
product 15 was obtained by reductive alkylation in the
presence of 35% formaldehyde and 10% Pd/C (Scheme
3). During elongation of the peptide chain, the yield of
each step was almost all above 90%, no N-carboxyanhy-
These a-halopyridinium-type coupling reagents can also be
used in solid-phase peptide synthesis, especially for the
synthesis of peptides containing N-methyl amino acid
residues. Using reagent BEP, the extremely hindered 8±11
tetrapeptide of Cyclosporin A was synthesized with the
Wang resin. The purity of the obtained crude peptide was
95% without any puri®cation after removal from the Wang
resin with 50% tri¯uoroacetic acid (Scheme 4).
dride (NCA) was detected although such by-products
would become dominant for halophosphonium type
reagents, such as PyBroP and PyCloP, mediated coupling
reactions in the case of Boc-protected amino acids as
carboxylic components.31
The ef®ciency of these a-halopyridinium salts can be further
proved by the successful total synthesis of the immuno-
suppressive cyclic undecapeptide Cyclosporin O in 18±
23% overall yield with the rationally combined utilization
of reagent BDMP and BEMT.32
For further determination of the widespread usefulness of
these 2-halopyridinium type reagents, we also synthesized

8126
P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
Figure 4. Proposed mechanism for a-halopyridinium salt mediated coupling reactions.
1
the linear undecapeptide CsO by the solid-phase method
using BEP and FEP, respectively. The C-terminal residue
Fmoc-Ala-OH was anchored onto the Wang resin using
DCC/HOAt/DMAP in CH2Cl2. Because the application of
the ninhydrin test for monitoring the coupling of N-methyl
amino acids is impossible, the double coupling strategy
(2£3 h) was adopted. To further reduce the racemization
during coupling, HOAt was added as an additive. Thus,
the reaction condition used in the coupling was: (3 equiv.
of Fmoc-AA-OH/DMF13 equiv. of coupling reagent13
equiv. of HOAt19 equiv. of DIEA)/1 equiv. of NH2-AA0-
Wang resin with a coupling time of 2£3 h. The cleavage of
the peptide from the resin was carried out using 50% TFA in
CH2Cl2 at low temperature (218 to 2158C). Unfortunately,
the purity of the obtained linear undecapeptide was no more
than 5% shown by ESI-MS spectrum although the results of
UV analysis33 during the Fmoc deprotection indicated that
the coupling yield of each step was above 90%. It is most
likely that the amide bonds between hindered amino acids
are prone to undergo hydrolysis in the strongly acidic
medium.34
analyzing by H NMR and IR, consists of 2-tert-butoxy-4-
isopropyl-l-5(4H)-oxazolone and a small amount of
symmetric anhydride of Boc-Val-OH, besides Boc-Val-
OH, Et3HN1X2 and the byproduct N-ethyl-2-pyridone II
released from 2-halopyridinium salts, compared to the
spectra of authentic compounds prepared in advance. No
NCA was detected during the reaction. However, it is
reasonable to speculate that the oxazolones and symmetric
anhydrides may not be the main active intermediates since
the reactivity of the two species is not high enough to cause
the coupling of Z-Gly-Phe-OH with Val-OCH3´HCl to be
accomplished within 2 min at 2108C in a dilute solution and
promote the coupling reactions between N-methyl or Ca,Ca-
dialkyl amino acids effectively with low racemization.
Therefore, we speculated that other more reactive inter-
mediates, such as (acyloxy)pyridinium salt and acyl halide,
might participate in the coupling reaction, but they were
very liable to decomposition and hardly detected. Because
acyl ¯uorides are insensitive to the attack of neutral nucleo-
phile, such as water and alcohol, than acyl bromides and
acyl chlorides, we carried out another model reaction by
treating the dichloromethane solution of the Fmoc-Val-
OH with FEP in the presence of NEt3 at 2108C, and
monitored by IR. The appearance of the characteristic
absorption of the carbonyl ¯uoride group was observed at
1845 cm21. This indicated the Fmoc-Val-F was generated
immediately when the base NEt3 was added. On exposing
A tentative mechanism for 2-halopyridinium salt mediated
coupling reactions was proposed and studied by H NMR
1
and IR. The model reaction was carried out in CDCl3 by
treating Boc-Val-OH with 2-halopyridinium salt in the
present of NEt3. It was indicated that the reaction mixture,

P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
8127
the reaction mixture to the air for 1 h, the intensity of the
absorption at 1845 cm21 decreased to approximately 60% of
the original value due to hydrolysis of Fmoc-Val-F.
Milwaukee, WI, and used without puri®cation. PyClU,
BTFFH, BOP-Cl and amino acid derivatives were prepared
according to literature methods.4,20,22 Amino acids were all
l-con®guration if not otherwise stated. Cbz-N-methyl
amino acids were synthesized by the procedure of
McDermott and Benoiton.35 Fmoc-N-methyl amino acids
were synthesized by the procedure of Freidinger et al.36
Based upon the above results, we speculated that the ®rst
step of carboxylic acid activation by 2-halopyridinium salts
involves the formation of an unstable acyloxypyridinium
salt intermediate I, in turn the intermediate reactes directly
with amino component to give the product or is com-
petitively converted into the acid halide which is subse-
quently converted into the dipeptide by aminolysis. A
small amount of 5(4H)-oxazolone and symmetric anhydride
of the carboxylic component are generated as minor active
intermediates during coupling, the former species is also
very labile to cause racemization due to its tautomerization
and enolization (Fig. 4). Comparing to halouronium and
halophosphonium type coupling reagents, the high reac-
tivity of a-halopyridinium slats may be attributed to the
instability of the pyridinium salts themselves and the higher
reactivity of the (acyloxy)pyridinium salts I than those of
(acyloxy)uronium and (acyloxy)phosphonium salts due to
the obvious electronic effect.
General procedure for the synthesis of pyridinium type
coupling reagents
To a solution of triethyloxonium tetra¯uoroborate or hexa-
chloroantimonate (10 mmol) in 10 mL ClCH2CH2Cl,
2-halogen pyridine (10 mmol) was added slowly under an
argon atmosphere. After stirring at room temperature for
1 h, and heating to 508C for a further 30 min. The reaction
mixture was cooled, diluted with anhydrous ether, ®ltered
and washed with ether. The crude product was crystallized
from acetone/ether to give corresponding pyridinium type
reagents as colorless crystals.
2-Bromo-1-ethyl pyridinium tetra¯uoroborate (BEP, 1).
BEP was synthesized from 2-bromopyridine and Et3O1BF42
according to the general procedure. Yield 95%, mp 103±
1048C; [Found: C, 30.58; H, 3.22; N, 4.96. C7H9BBrF4N
requires C, 30.70; H, 3.31; N, 5.11%]; nmax(KBr) 3106w,
1617s, 1571m, 1500m, 1467m, 1296m, 1050vs, 786s, 718w,
Conclusions
The 2-halopyridinium type coupling reagents, which can be
readily prepared from 2-halopyridine in one step as shelf-
stable crystals, demonstrated very high reactivity, low
racemization and excellent yields in peptide synthesis
both in solution and the solid-phase, especially the synthesis
of hindered peptides containing N-methyl amino acid
residues.
1
521w; H NMR (300 MHz, d6-acetone): dˆ1.69 (t, Jˆ
7.3 Hz, 3H, CH2CH3), 5.01 (q, Jˆ7.3 Hz, 2H, CH2CH3),
8.24 (m, 1H, 5-CH-Py), 8.53±8.57 (m, 2H, 3,4-CH-Py),
9.32 (d, Jˆ6.9 Hz, 1H, 6-CH-Py); FAB-MS m/z: 186
[M2BF24 ]1, 188 [M2BF24 12]1.
2-Fluoro-1-ethyl pyridinium tetra¯uoroborate (FEP, 2).
FEP was synthesized from 2-¯uoropyridine and Et3O1BF42
according to the general procedure. Yield 95%; mp 51±
528C; [Found: C, 39.32; H, 4.17; N, 6.54. C7H9BF5N
requires C, 39.48; H, 4.26; N, 6.58%]; nmax(KBr) 3039w,
1643s, 1587m, 1519s, 1475m, 1307m, 1070vs, 847m, 786s,
Experimental
Melting points were determined in capillary tubes and are
uncorrected. IR spectra were measured with Schimadazu
IR-440 spectrometer. NMR spectra were recorded on
Bruker AM 300 or Bruker DRX 400 instruments. Chemical
shifts are reported as ppm (d units) down®eld from a tetra-
methylsilane internal standard. Because in most cases the
spectra demonstrated multiple conformations, generally
with different coupling constants for the different rotamers,
J values are for the most part omitted. Mass spectra were
taken with HP5890A, and VG QUATTRO mass spec-
trometers. Elemental analyses for carbon, hydrogen and
nitrogen were determined on a MOD-1106 elemental
analyzer. Optical rotations were recorded with a Perkin±
Elmer 241 MC polarimeter. HPLC analyses were carried
out on a waters or Varian-SY-5000 instrument and using
either a Kromasil RP-18 (5£250 mm) or a Waters mBonda-
pak C18 (4.6£300 mm) column. Flash column chroma-
tography was performed with 300±400 meshes silica gel,
and analytical thin layer chromatography was performed on
precoated silica gel plates (GF-254) with the systems (v/v)
indicated. Solvents and reagents were puri®ed by standard
methods as necessary. The 60±908C Petroleum (PE) ether
was used.
1
725w, 522w; H NMR (300 MHz, d6-acetone): dˆ1.69 (t,
Jˆ7.3 Hz, 3H, CH2CH3), 4.85 (q, Jˆ7.3 Hz, 2H, CH2CH3),
8.02±9.11 (m, 4H, aryl); 19F NMR (60 MHz, d6-acetone,
CF3COOH): dˆ70.2 (s, 1F); FAB-MS m/z: 126
[M2BF24 ]1.
2-Bromo-1-ethyl pyridinium hexachloroantimonate
(BEPH, 3). BEPH was synthesized from 2-bromopyridine
and Et3O1SbCl26 according to the general procedure. Yield
90%, mp 240±2428C; [Found: C, 16.01; H, 1.80; N, 2.87.
C7H9BrCl6NSb requires C, 16.12; H, 1.74; N, 2.69%];
n
max(KBr) 3133w, 2987w, 1614m, 1546s, 1465m, 1407m,
1
1212s, 1130s, 1053m, 741s, 630m; H NMR (300 MHz,
d6-acetone): dˆ1.68 (t, Jˆ7.2 Hz, 3H, CH2CH3), 4.99 (q,
Jˆ7 Hz, 2H, CH2CH3), 8.22 (m, 1H, 5-CH-Py), 8.51±8.58
(m, 2H, 3,4-CH-Py), 9.29 (d, Jˆ6.5 Hz, 1H, 6-CH-Py).
2-Fluoro-1-ethyl pyridinium hexachloroantimonate
(FEPH, 4) FEPH was synthesized from 2-¯uoropyridine
and Et3O1SbCl26 according to the general procedure.
Yield 91%, mp 262±2468C; [Found: C, 18.09; H, 1.80; N,
3.02. C7H9Cl6NFSb requires C, 18.25; H, 1.97; N, 3.04%];
nmax (KBr) 3088w, 2995w, 1639vs, 1586s, 1516s, 1474m,
1310m, 1149m, 1091m, 847m, 776s, 728w; 1H NMR
DCC, PyBroP, HOAt, 2-bromo pyridine and 2-¯uoro
pyridine were purchased from Aldrich Chemical Co. of

8128
P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
(300 MHz, d6-acetone): dˆ1.75 (t, Jˆ7.2 Hz, 3H,
CH2CH3), 4.93 (q, Jˆ7.2 Hz, 2H, CH2CH3), 8.15±9.15
(m, 4H, aryl); 19F NMR (300 MHz, d6-acetone, CF3COOH):
dˆ68.8 (s, 1F).
FEP (0.117 g, 0.55 mmol) in 2 mL CH2Cl2, DIEA
(0.28 mL, 1.60 mmol) was added. The reaction mixture
was stirred at room temperature for 4 h. Yield: 0.234 g
(95%), [a]2D0ˆ2135.6 (c 1, MeOH), Rf 0.66 (AcOEt/PE:
1/3); 1H NMR (300 MHz, CDCl3) four conformers:
dˆ0.46±1.06 (m, 12H, CH3 of MeLeu, MeVal), 1.10±
1.79 (m, 3H, g-CH-MeLeu, b-CH2-MeLeu), 1.90±2.26
(m, 1H, b-CH-MeVal), 2.65±2.98 (m, 6H, N-CH3 of
MeLeu, MeVal), 3.41, 3.63, 3.65, 3.70 (4s, 3H, OCH3),
4.12±5.18 (m, 5H, 9-CH-Fluorenyl, CH2-Fmoc, a-CH-
MeVal, a-CH-MeLeu), 7.28±7.47 (m, 4H, 2,3,6,7-CH-
Fluorenyl), 7.50±7.69 (m, 2H, 1,8-CH-Fluorenyl), 7.70±
7.85 (m, 2H, 4,5-CH-Fluorenyl); EIMS m/z: 495
[M1H]1, 463 [M2OCH3]1, 322 [(M2MeVal-OCH3]1,
179 [Fmoc-CO2]1.
HPLC monitoring of model reactions for the evaluation
of the ef®ciency of halogenated coupling reagents with
the model reaction Z-Gly-Phe-OH1Val-OMe´HCl!
Z-Gly-d/l-Phe-Val-OMe
In the presence of DIEA (78 mL, 0.448 mmol), Z-Gly-Phe-
OH (50 mg, 0.14 mmol) and Val-Ome´HCl (26 mg, 0.154
mmol) were coupled with the tested reagent (0.154 mmol)
in CH2Cl2 (1.5 mL) at 2108C. Boc-Phe-Val-OMe (66 mg,
0.18 mmol) was added as the internal reference. Aliquots
(10 mL) from the reaction mixture were quenched and
dissolved in 100 mL buffer solution (CH3OH/H2O/TFA:
50/50/1). The resultant samples were analyzed by HPLC
to give the following results: Z-Gly-Phe-OH (tRˆ
4.04 min); Z-Gly-l-Phe-Val-OMe (tRˆ9.24 min); Z-Gly-d-
Phe-Val-OMe (tRˆ10.28 min); Boc-Phe-Val-OMe (tRˆ
15.82 min) by comparing to the prepared reference
compounds. Peak areas were compared in order to obtain
the chemical yields (yield (%)ˆ[(LL/X11DL/X2)/
aS]£100%). The percentage of epimers was calculated
according to the equation: D (%)ˆ[DL/X2/(LL/X11DL/
X2)]£100%; where LL refers to the area of Z-Gly-l-Phe-
Val-OMe, DL refers to that of Z-Gly-d-Phe-Val-OMe, S
refers to that of Boc-Phe-Val-OMe, aˆ0.778 which is the
molar ratio between the Z-Gly-Phe-OH and Boc-Phe-Val-
OMe, X1ˆ1.269 and X2ˆ1.254 which are the determined
correction factors for absorption difference (220 nm)
between the references.
N-(tert-Butyloxycarbonyl)-a-aminoisobutyric acid penta-
¯uorophenyl ester (Boc-Aib-OPfp). To a cold solution of
Boc-Aib-OH (0.305 g, 1.50 mmol), KOPfp (0.333 g,
1.50 mmol) and FEP (0.352 g, 1.65 mmol) in 3 mL
CH2Cl2, DIEA (0.29 mL, 1.65 mmol) was added. The reac-
tion mixture was stirred at room temperature for 1 h. Yield:
0.419 g (76%), mp 105±1068C, Rf 0.70 (AcOEt/PE: 1/12);
n
max(KBr) 3389s, 2995m, 1772s, 1705s, 1521s, 1454m,
1388m, 1292m, 1258m, 1169m, 1080s, 995s; 1H NMR
(90 MHz, CDCl3): dˆ1.51 (s, 9H, 3CH3-But), 1.68 (s, 6H,
2b-CH3-Aib), 5.06 (br, 1H, NH): 19F NMR (60 MHz,
CDCl3, CF3COOH): dˆ273.9 (d, Jˆ20 Hz, 2F), 279.5
(t, Jˆ20 Hz, 1F), 284.0 (t, Jˆ20 Hz, 2F); EIMS m/z: 370
[M1H]1, 57 [But]1.
Synthesis of the 8±11 protected tetrapeptide fragment of
Cyclosporin A using reagent BEP N-[(9H-Fluoren-9-
ylmethoxy)carbonyl]-N-methylleucyl-N-methylvaline
benzyl ester (Fmoc-MeLeu-MeVal-OBzl, 5). To a cold
solution of Fmoc-MeLeu-OH (0.143 g, 0.389 mmol),
MeVal-OBzl´TFA (0.137 g, 0.409 mmol) and BEP
(0.117 g, 0.428 mmol) in 2 mL CH2Cl2, DIEA (0.21 mL,
1.23 mmol) was added. The reaction mixture was stirred
at room temperature for 4 h. Yield: 0.203 g (91%), Rf 0.53
HPLC conditions: Column: Kromasil KR 100±10 C18
(4.6£25 cm). Eluant: 48% CH3CN (0.1% TFA). Flow
rate: 1.5 mL/min. Detection: 220 nm (0.5 AUFS).
Illustrations of the preparation of oligopeptides and
esters using 2-halopyridinium salts N-[(9H-Fluoren-9-
ylmethoxy)carbonyl]-valyl-N-methyl-valine methyl ester
(Fmoc-Val-MeVal-OCH3). To a cold solution of Fmoc-
Val-OH (0.187 g, 0.55 mmol), MeVal-OCH3´HCl (91 mg,
0.50 mmol) and BEP (0.151 g, 0.55 mmol) in 2 mL
CH2Cl2, DIEA (0.28 mL, 1.6 mmol) was added. The reac-
tion mixture was stirred at room temperature for 1 h. Yield:
0.206 g (88%), [a]D23ˆ286.0 (c 0.3, CHCl3), Rf 0.65
1
(AcOEt/PE: 1/4), [a]2D2ˆ2122 (c 0.2, CHCl3); H NMR
(300 MHz, CDCl3) four conformers: dˆ0.39±0.95 (m,
12H, CH3 of MeLeu, MeVal), 1.16±1.63 (m, 3H, g-CH-
MeLeu, b-CH2-MeLeu), 1.97±2.23 (m, 1H, b-CH-
MeVal), 2.55±2.88 (m, 6H, N-CH3 of MeLeu, MeVal),
3.94±5.23 (m, 7H, 9-CH-Fluorenyl, CH2-Fmoc, a-CH-
MeVal, a-CH-MeLeu, CH2-OBzl), 7.01±7.74 (m, 13H,
aryl); EIMS m/z: 571 [M1H]1, 555 [M2CH3]1, 463
[M2OBzl]1, 322 [M2MeVal-OBzl-CO]1, 179 [Fmoc-
CO2]1, 91 [PhCH2]1.
1
(AcOEt/PE: 1/1); H NMR (300 MHz, CDCl3) two con-
formers: dˆ0.58±1.04 (m, 12H, CH3 of Val, MeVal),
2.02±2.38 (2m, 2H, b-CH of Val, MeVal), 2.91, 3.07 (2s
1: 7, 3H, N-CH3-MeVal), 3.71 (s, 3H, OCH3), 4.07±5.02 (m,
5H, a-CH-MeVal, 9-CH-Fluorenyl, CH2-Fmoc, a-CH-Val),
5.55 (d, Jˆ9.3 Hz, 1H, NH-Val), 7.32 (t, Jˆ7.4 Hz, 2H, 2,
7-CH-Fluorenyl), 7.41 (t, Jˆ7.4 Hz, 2H, 3, 6-CH-Fluore-
nyl), 7.60 (d, Jˆ7.3 Hz, 2H, 1,8-CH-Fluorenly), 7.77 (d,
Jˆ7.4 Hz, 2H, 4,5-CH-Fluorenyl); EIMS m/z: 467
[M1H]1, 294 [M2MeVal-OCH3±CO]1, 179 [Fmoc-
CO2]1.
N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-methylleucyl-
N-methylleucyl-N-methyl valine benzyl ester (Fmoc-
MeLeu-MeLeu-MeVal-OBzl, 6). Fmoc-MeLeu-MeVal-
OBzl (0.148 g, 0.259 mmol) was dissolved in CH3CN
(2 mL) and treated with an equal volume of diethylamine
under nitrogen atmosphere until TLC analysis indicated that
the starting material disappeared (ca. 40 min). The solution
was concentrated in vacuo, the residue was dissolved in
CH3CN and concentrated again to give H-MeLeu-MeVal-
OBzl, which was further dried in vacuo for 2 h and precipi-
tated the following coupling reaction without puri®cation.
N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-methyl-leucyl-
N-methyl-valine methyl ester (Fmoc-MeLeu-MeVal-
OCH3). To a cold solution of Fmoc-MeLeu-OH (0.184 g,
0.50 mmol), MeVal-OCH3´HCl (0.100 g, 0.55 mmol) and
To
a cold solution of Fmoc-MeLeu-OH (0.114 g,

P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
8129
0.311 mmol), BEP (85 mg, 0.311 mmol) and H-MeLeu-
MeVal-OBzl (0.259 mmol) in 2 mL CH2Cl2, DIEA
(108 mL, 0.622 mmol) was added. The reaction mixture
was stirred at room temperature for 4 h. Yield: 87 mg
(48%), Rf 0.41 (AcOEt/PE: 1/4), [a]2D0ˆ2114.8 (c 1,
CH3OH); 1H NMR (300 MHz, CDCl3) more than two
conformers: dˆ0.51±1.01 (m, 18H, CH3 of 2MeLeu,
MeVal), 1.25±1.78 (m, 6H, 2b-CH2-MeLeu, 2g-CH-
MeLeu), 2.19 (m, 1H, b-CH-MeVal), 2.64±2.91 (m, 9H,
N-CH3 of 2MeLeu, MeVal), 4.17±5.49 (m, 8H, 9-CH-
Fluorenyl, CH2-Fmoc, a-CH-MeVal, CH2-OBzl, 2a-CH-
MeLeu), 7.26±7.48 (m, 9H, 2, 3, 6, 7-CH-Fluorenyl,
Ph-OBzl), 7.50±7.63 (m, 2H, 1,8-CH-Fluorenyl), 7.65±
7.84 (m, 2H, 4,5-CH-Fluorenyl); EIMS m/z: 697 M1, 477
[M2MeVal-OBzl]1, 322 [(M2MeLeu-MeVal-OBzl-
CO]1, 179 [(Fmoc-CO2]1, 91 [PhCH2]1.
(94.7%), mp 158±1608C, Rf 0.31 (AcOEt/MeOH: 1/1);
EIMS m/z: 303 [M1H]1, 91 [PhCH2]1.
N-(tert-Butyloxycarbonyl)-valyl-methylvaline
methyl
ester (Boc-Val-MeVal-OCH3, 10). To a cold solution of
Boc-Val-OH (0.782 g, 3.6 mmol), MeVal-OCH3´HCl
(0.545 g, 3.0 mmol) and FEP (0.767 g, 3.6 mmol) in 5 mL
CH2Cl2, DIEA (1.78 mL, 10.2 mmol) was added. The reac-
tion mixture was stirred at room temperature for 2 h. Yield:
0.951 g (92%), Rf 0.56 (AcOEt/PE: 1/4), [a]2D0ˆ2136.0 (c
1
1, MeOH); H NMR (90 MHz, CDCl3) two conformers:
dˆ0.75±1.11 (m, 12H, CH3 of Val, MeVal), 1.44 (s, 9H,
But), 1.86±2.35 (2m, 2H, b-CH of Val, MeVal), 2.87, 3.08
(2s, 3H, N-CH3-MeVal), 3.70 (s, 3H, OCH3), 4.08±4.64
(2m, 2H, a-CH of Val, MeVal), 4.93, 5.25 (2d, Jˆ10 Hz,
1H, NH-Val); EI-MS m/z: 345 [M1H]1, 344 M1, 57[But]1.
N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-d-alanyl-N-methyl-
leucyl-N-methylleucyl-N-methylvaline benzyl ester
(Fmoc-d-Ala-MeLeu-MeLeu-MeVal-OBzl, 7). Fmoc-
MeLeu-MeLeu-MeVal-OBzl (0.080 g, 0.115 mmol) was
deprotected according to above experimental procedure to
give H-MeLeu-MeLeu-MeVal-OBzl, which was further
dried in vacuo for 2 h and precipitated the following
coupling reaction without puri®cation. To a cold solution
of Fmoc-d-Ala-OH (54 mg, 0.172 mmol), BEP (47 mg,
0.172 mmol) and H-MeLeu-MeLeu-MeVal-OBzl (0.115
mmol) in 1 mL CH2Cl2, DIEA (60 mL, 0.344 mmol) was
added. The reaction mixture was stirred at room temperature
overnight. Yield: 83 mg (94%), Rf 0.53 (AcOEt/PE: 1/2),
[a]2D5ˆ2145.4 (c 0.5, CHCl3); 1H NMR (300 MHz, CDCl3)
two conformers: dˆ0.78±1.96 (m, 27H, CH3 of D-Ala,
2MeLeu, MeVal, 2b-CH2-MeLeu, 2g-CH-MeLeu), 2.18
(m, 1H, b-CH-MeVal), 2.52±3.04 (m, 9H, N-CH3 of
2MeLeu, MeVal), 4.19±4.44 (m, 3H, 9-CH-Fluorenyl,
CH2-Fmoc), 4.67 (m, 1H, a-CH-d-Ala), 4.88 (d, Jˆ
10.5 Hz, 1H, a-CH-MeVal), 5.14 (m, 2H, CH2-OBzl),
5.37±5.54 (m, 2H, 2a-CH-MeLeu), 5.76 (m, 1H, NH-d-
Ala), 7.24±7.45 (m, 9H, 2, 3, 6, 7-CH-Fluorenyl, Ph-
OBzl), 7.56 (d, Jˆ7.3 Hz, 2H, 1,8-CH-Fluorenyl), 7.76 (d,
Jˆ7.4 Hz, 2H, 4,5-CH-Fluorenyl); EI-MS m/z: 768 M1, 548
[M2MeVal-OBzl]1, 422 [M2MeLeu-MeVal-OBzl]1, 91
[PhCH2]1.
Valyl-N-methylvaline methyl ester hydrochloride (HCl´
Val-MeVal-OCH3, 11). Boc-Val-MeVal-OCH3 (0.812 g,
2.36 mol) was deprotected with 4N HCl/AcOEt (8 mL).
Yield: 0.605 g (91.4%), mp 181±1828C, Rf 0.48 (AcOEt/
MeOH: 4/1).
N-(tert-Butyloxycarbonyl)-valyl-valyl-methylvaline methyl
ester (Boc-Val1-Val2-MeVal3-OCH3, 12). To a cold
solution of Boc-Val-OH (83 mg, 0.38 mmol), Val-MeVal-
OCH3´HCl (97 mg, 0.35 mmol) and BEP (104 mg,
0.38 mmol) in 1 mL CH2Cl2, DIEA (0.19 mL, 1.11 mmol)
was added. The reaction mixture was stirred at room
temperature for 1 h. Yield: 0.15 g (97%), mp: 138±1398C,
1
Rf 0.43 (AcOEt/PE: 1/4); H NMR (300 MHz, d6-acetone)
one main conformers: dˆ0.79±1.15 (m, 18H, CH3 of Val1,
Val2, MeVal), 1.41 (s, 9H, But), 1.95±2.28 (3m, 3H, b-CH
of Val1, Val2, MeVal), 3.11 (s, 3H, N-CH3-MeVal), 3.68 (s,
3H, OCH3), 4.03 (m, 1H, a-CH-MeVal), 4.70 (m, 1H,
a-CH-Val2), 4.86 (d, Jˆ10.7 Hz, 1H, a-CH-Val1), 6.08,
7.57 (2d, Jˆ8.5 Hz, 2H, NH of Val1, Val2); EI-MS m/z:
444 [M1H]1, 412 [M2OCH3]1, 57 [But]1.
N-(tert-Butyloxycarbonyl)-valyl-valyl-methylvaline (Boc-
Val1-Val2-MeVal3-OH, 13). Boc-Val-Val-MeVal-OCH3
(0.855 g, 1.93 mmol) was saponi®ed with 2N NaOH to
give product. Yield: 0.812 g (98%), mp 72±748C, Rf 0.76
(AcOEt/MeOH: 1/1); EI-MS m/z: 430 [M1H]1, 412
[M2H2O]1, 329 [M1H2Boc]1, 57[But]1.
Synthesis of the pentapeptide moiety of Dolastatin 15
using pyridinium type coupling reagents N-(tert-Butyl-
oxycarbonyl)-prolyl-proline benzyl ester (Boc-Pro-Pro-
OBzl, 8). To a cold solution of Boc-Pro-OH (0.431 g,
2.0 mmol), Pro-OBzl´TFA (0.702 g, 2.2 mmol) and BEP
(0.603 g, 2.2 mmol) in 3 mL CH2Cl2, DIEA (1.1 mL,
6.4 mmol) was added. The reaction mixture was stirred at
room temperature for 1 h. Yield: 0.726 g (91%), [a]2D3ˆ
2109.4 (c 1, CHCl3), Rf 0.26 (AcOEt/PE: 1/2);1H NMR
(90 MHz, CDCl3) two conformers: dˆ1.44 (s, 9H, But),
1.69±2.33 (m, 8H, 2b-CH2-Pro, 2g-CH2-Pro), 3.31±3.88
(m, 4H, 2d-CH2-Pro), 4.05±4.52 (2m, 2H, 2a-CH-Pro),
4.91±5.10 (m, 2H,CH2-OBzl), 7.32 (s, 5H, Ph-OBzl); EI-
MS m/z: 403 [M1H]1, 402 M1, 302 [M1H2Boc]1, 91
[PhCH2]1, 57 [But]1.
N-(tert-Butyloxycarbonyl)-valyl-valyl-methylvalyl-prolyl-
proline benzyl ester (Boc-Val1-Val2-MeVal3-Pro4-Pro5-
OBzl, 14). To a cold solution of Boc-Val-MeVal-OH
(0.652 g, 1.52 mmol), Pro-Pro-OBzl´HCl (0.540 g,
1.59 mmol) and BEP (0.458 g, 1.67 mmol) in 7 mL
CH2Cl2, DIEA (0.83 mL, 4.78 mmol) was added at 08C.
The reaction mixture was stirred at room temperature for
2 h. Yield: 0.949 g (88%), mp 94±968C, [a]2D3ˆ2181 (c
0.3, CHCl3); [Found: C, 63.08; H, 8.38; N, 9.54.
C38H59N5O8´0.5H2O requires C, 63.13; H, 8.37; N,
9.69%]; nmax(KBr) 3319s, 2967s, 2877sh, 1746sh, 1716sh,
1
1663sh, 1634vs, 1499s, 1442s, 1171s, 1094w, 699w; H
NMR (300 MHz, d6-acetone): dˆ0.73±1.01 (m, 18H, CH3
of Val1, Val2, MeVal3), 1.42 (s, 9H, But), 1.72±2.31 (3m,
11H, b-CH of Val1, Val2, MeVal3, 2b-CH2-Pro, 2g-CH2-
Pro), 3.13 (s, 3H, N-CH3-MeVal3), 3.56±3.69 (m, 2H,
d-CH2-Pro), 3.75±3.87 (m, 2H, d-CH2-Pro), 4.05 (m, 1H,
Prolyl-proline benzyl ester hydrochloride (HCl´Pro-Pro-
OBzl, 9). Boc-Pro-Pro-OBzl (0.825 g, 2.05 mol) was depro-
tected with 4N HCl/AcOEt (8 mL) at 08C. Yield: 0.658 g

8130
P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
a-CH-MeVal3), 4.48 (dd, J1ˆ4.2 Hz, J2ˆ8.7 Hz, 1H,
a-CH-Pro), 4.63 (dd, J1ˆ4.6 Hz, J2ˆ8.9 Hz, 1H, a-CH-
Pro), 4.75 (d, Jˆ7.9 Hz, 1H, a-CH-Val2), 5.10 (d, Jˆ
10.9 Hz, 1H, a-CH-Val1), 5.07, 5.19 (AB, Jˆ12.5 Hz, 2H,
CH2-OBzl), 6.20 (d, Jˆ10 Hz, 1H, NH-Val2), 7.35±7.40
(m, 5H, Ph-OBzl), 7.48 (d, Jˆ8 Hz, 1H, NH-Val1);
ESI-MS m/z: 1450 [M1M1Na]1, 753 [M1K]1, 737
[M1Na]1, 715 [M1H]1, 546 [(M1H1K)/2]1, 538
[(M1H1Na)/2]1.
mmol) for 30 min. The resin was ®ltered and washed with
DMF (10£, each with 3 mL for 1 min). Fmoc-protected
amino acid (3 equiv.), 2-halopyridinium salt (3 equiv.)
HOAt (3 equiv.) in DMF (2 mL) was added to the resin,
then DIEA (9 equiv.) was added and the resin mixutre
was shaken for 3 h. The coupling reaction was performed
twice.
The peptide was cleaved from the resin using 50% TFA at
low temperature. The ¯ask containing the above resin was
chilled under an argon atmosphere in an ethylene glycol/
CO2 bath held at 215 to 2208C. TFA (8 mL) which was
precooled in the same bath, was added to the ¯ask, the
reaction mixutre was stirred at the low temperature for 3
h. The TFA was removed at 2158C by distillation into a
CO2/acetone trap under vacuum, and then the residue was
treated with AcOEt (5 mL) and concentrated repeatedly in
vacuo. The residue was washed with AcOEt (200 mL). The
AcOEt solution was concentrated, dried and evaluated the
purity of the crude peptide by HPLC and ESIMS. The crude
linear undecapeptide of CsO synthesized using BEP and
FEP was evaluated by ESI-MS and showed no more than
5% purity.
N,N-Dimethyl-valyl-valyl-methylvalyl-prolyl-proline
(Me2Val1-Val2-MeVal3-Pro4-Pro5-OH, 15). Boc-Val-Val-
MeVal-Pro-Pro-OBzl (35 mg, 0.049 mmol) was depro-
tected by 50% TFA in 2 mL CH2Cl2 to give TFA´H-Val-
Val-MeVal-Pro-Pro-OBzl as white solid, which was
dissolved in 2.5 mL methanol and reductively alkylated in
the presence of 10% Pd/C (80 mg), 37% formaldehyde
aqueous solution (0.32 mL, 3.92 mmol) and NEt3 (7 mL,
0.049 mmol) at room temperature. Another part of 35%
formaldehyde solution (0.15 mL) was added after 24 h and
the reaction mixture was further hydrogenated for 48 h.
Yield: 22 mg (82%), Rf 0.27 (MeOH); nmax(KBr) 3400s,
2972s, 1730sh, 1673vs, 1639vs, 1473m, 1291w, 1202s,
1137sh, 799w, 721w; 1H NMR (400 MHz, acetone-d6):
dˆ0.76±1.12 (m, 18H, CH3 of Me2Val1, Val2, MeVal3),
1.81±2.37 (m, 11H, b-CH of Me2Val1, Val2, MeVal3,
2b-CH2-Pro, 2g-CH2-Pro), 2.98 (s, 6H, 2N-CH3-Me2Val1),
3.19 (s, 3H, N-CH3-MeVal3), 3.57±3.73 (m, 2H, d-CH2-
Pro), 3.75±3.92 (m, 2H, d-CH2-Pro), 3.95 (d, Jˆ8.5 Hz,
1H, a-CH-MeVal3), 3.61±4.05 (br, 1H, COOH), 4.45 (m,
1H, a-CH-Pro), 4.66 (dd, J1ˆ5.1 Hz, J2ˆ8.5 Hz, 1H, a-CH-
Pro), 4.75 (d, Jˆ8.0 Hz, 1H, a-CH-Val2), 5.10 (d, Jˆ
11.0 Hz, 1H, a-CH-Me2Val1); ESI-MS m/z: 1104.6
[M1M1H]1, 575.1 [M1Na]1, 552.5 [M1H]1.
HPLC analysis indicated the purifty of Fmoc-d-Ala-
MeLeu-MeLeu-MeVal-OH synthesized by BEP was 95%.
The overall yield of the tetrapeptide was 85%. tRˆ10.7 min
(HPLC conditions: Column: Kromasil KR 100±10 C18
(4.6£25 cm). Eluant: 48% CH3CN (0.1% TFA). Flow
rate: 1.5 mL/min. Detection: 220 nm (0.5 AUFS)). After
puri®cation, the ®nal product characterized and proved to
be the desired product Fmoc-d-Ala-MeLeu-MeLeu-MeVal-
OH. Mp 80±828C, Rf 0.62 (AcOEt/CH3OHˆ10/1), [a]2D0ˆ
2102.0 (c 0.25, CHCl3); [Found: C, 65.66; H, 8.01; N, 7.96.
C38H54N4O7´H2O requires C, 65.49; H, 8.10; N, 8.04%];
n
max(KBr) 3316w, 2960s, 1726s, 1643s, 1451m, 1247m,
Synthesis of the 8±11 fragment of CsA and the total
synthesis of linear undecapeptide of CsO in the solid-
phase using pyridinium type reagent
1
1063m, 741w; H NMR (300 MHz, CDCl3): dˆ0.76±1.20
(m, 21H, CH3 of d-Ala, Leu, Leu, Val), 1.24±1.91 (m, 6H,
2b-CH2-Leu, 2g-CH-Leu), 2.32 (m, 1H, b-CH-Val), 2.77±
3.90 (m, 9H, N-CH3 of 2Leu, Val), 4.06±4.49 (m, 4H,
9-CH-Fluorenyl, CH2-Fmoc, a-CH-Val), 4.72 (m, 1H,
a-CH-d-Ala), 5.44±5.56 (m, 2H, 2a-CH-Leu), 5.92, 6.60
(2d, Jˆ8.0 Hz, 1H, NH-d-Ala), 7.31 (m, 2H, 2,7-CH-Fluor-
enyl), 7.40 (t, Jˆ7.3 Hz, 2H, 3, 6-CH-Fluorenyl), 7.60 (m,
2H, 1,8-CH-Fluorenyl), 7.76 (d, Jˆ7.6 Hz, 2H, 4,5-CH-
Fluorenyl); EI-MS m/z: 661 [M2OH]1, 421 [M2MeLeu-
MeVal-OH]1, 179 [Fmoc-CO2]1.
Anchoring of Fmoc-Ala-OH on Wang Resin (general
procedure). To a solution of Fmoc-Ala-OH (1.33 g,
4.27 mmol), HOAt (0.174 g, 1.28 mmol), DMAP (0.104 g,
0.854 mmol) and DCC (0.881 g, 4.27 mmol) in CH2Cl2/
DMF (4/1, 25 mL), Wang resin (2.0 g, 0.854 mmol) was
added at 08C. The mixture was gently stirred 16 h at room
temperature. The resin was ®ltered, washed successively
with DMF (3£), MeOH (3£), DMF/H2O 9/1 (v/v, 3£),
DMF (3£) and MeOH (3£), and dried under vacuum. UV
and HPLC analysis indicated 84% substitution of the resin.
The unreacted hydroxyl group was capped using acetic
anhydride.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China.
Anchoring of Fmoc-MeVal-OH on Wang Resin. Fmoc-
MeVal-OH was anchored onto Wang resin according to
above general procedure except the reaction time was
prolonged to 30 h to afford 85% substitution of the resin.
References
General procedure for the solid-phase peptide synthesis
using 2-halopyridinium salts
1. Castro, B.; Dromoy, J. R.; Evin, G.; Selve, C. Tetrahedron Lett.
1975, 1219.
2. Dourtoglou, V.; Ziegler, J. C.; Gross, B. Tetrahedron Lett.
1978, 1269.
Starting from Fmoc-Ala-Wang resin (0.3 g, 0.107 mmol) or
Fmoc-MeVal-Wang resin (0.4 g, 0.145 mmol) Fmoc depro-
tection was performed with 20% piperidine in DMF (20 mL/
3. Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31,
205.

P. Li, J.-C. Xu / Tetrahedron 56 (2000) 8119±8131
4. (a) Chen, S. Q.; Xu, J. C. Tetrahedron Lett. 1992, 33, 647. 20. El-Faham, A. Chem. Lett. 1998, 671.
8131
(b) Coste, J.; Frerot, E.; Jouin, P. Tetrahedron Lett. 1991, 32, 1967.
5. Carpino, L. A.; El-Faham, A.; Albericio, F. Tetrahedron Lett.
1994, 35, 2279.
21. Przybylski, J.; Wasilewski, K.; Legowska, A. Pol. PL 170331
B1, ®led 10/30/92, published 11/29/96, assigned to Uniwersytet
Gdanski, Pol., 6 pp (Poland).
6. Ehrlich, A.; Rothemund, S.; Brudel, M.; Beyermann, M.;
Carpino, L. A.; Bienert, M. Tetrahedron Lett. 1993, 34, 4781.
7. Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F.
J. Chem., Soc. Chem. Commun. 1994, 201.
22. (a) Van der Auwera, C.; Anteunis, M. J.O. Int. J. Pept. Protein
Res. 1987, 29, 574. (b) Van der Auwera, C.; Anteunis, M. O. Bull.
Soc. Chim. Belg. 1986, 95, 203.
23. Li, P.; Xu, J. C. Tetrahedron Lett. 1999, 40, 8301.
24. Mukaiyama, T.; Usui, M.; Shimada, E. Chem. Lett. 1975,
1045.
8. Alsina, J.; Giralt, E.; Albericio, F. Tetrahedron Lett. 1996, 37,
4195.
9. (a) Li, P.; Xu, J. C. Tetrahedron Lett. 1999, 40, 3605. (b) Li, P.;
Xu, J. C. J. Pept. Res. 2000, 55, 110.
10. Li, P.; Xu, J. C. Chem. Lett. 1999, 1163.
25. Mukaiyama, T.; Usui, M.; Saigo, K. Chem. Lett. 1976, 49.
26. Bald, E.; Saigo, K.; Mukaiyama, T. Chem. Lett. 1975, 1163.
27. Barlin, G. B.; Benbow, J. A. J. Chem. Soc., Perkin Trans. 2
1974, 790.
11. Li, P.; Xu, J. C. Tetrahedron Lett. 2000, 41, 721.
12. (a) Sheehan, J. C.; Hess, G. P. J. Am. Chem. Soc. 1955, 77,
1067. (b) Sarantakis, D.; Teichman, J.; Lien, E. L.; Fenichel, R.
Biochem. Biophys. Res. Commun. 1976, 73, 336. (c) Shechan, J. C.;
Cruickshank, P. A.; Boshart, G. L. J. Org. Chem. 1961, 26, 2525.
13. (a) Wieland, T.; Schaefer, W. Angew. Chem. 1951, 63, 146.
(b) Bodanszky, M. Nature 1955, 175, 685.
28. Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
29. Wenger, R. M. Helv. Chim. Acta. 1983, 66, 2672.
30. Pettit, G. R.; Kamano, Y.; Dufresne, C.; Cerny, R. L.; Herald,
C. L.; Schmidt, J. M. J. Org. Chem. 1989, 54, 6005.
31. Frerot, E.; Coste, J.; Poncet, J.; Jouin, P. Tetrahedron Lett.
1992, 33, 2815.
14. Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97,
2243.
32. Li, P.; Xu, J. C. J. Org. Chem. 2000, 65, 2951.
33. (a) For quantitation of Fmoc protected petide segment on
resin, UV absorbance of the fulvene-piperidine adduct at 301 nm
was measured after cleavage of the Fmoc group with piperidine.
(b) Meienhofer, J.; Waki, M.; Heimer, E.; Lambros, T. J.;
Makofske, R.; Chang, C.-D. Int. J. Pept. Protein Res. 1979, 13, 35.
34. Albericio, F.; Cases, M.; Alsina, J.; Triolo, S. A.; Carpino,
L. A.; Kates, S. A. Tetrahedron Lett. 1997, 38, 4853 (and
references cited therein).
15. (a) Albericio, F.; Bo®ll, J. M.; El-Faham, A.; Kates, S. A.
J. Org. Chem. 1998, 63, 9678. (b) Story, S. C.; Aldrich, J. V. Int.
J. Pept. Protein Res. 1994, 43, 292.
16. Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M.-H.; Jouin, P.
Tetrahedron 1991, 47, 259.
17. Coste, J.; Dufour, M.-N.; Pantaloni, A.; Castro, B.
Tetrahedron Lett. 1990, 31, 669.
18. Akaji, K.; Kuriyama, N.; Kiso, Y. Tetrahedron Lett. 1994, 35,
3315.
35. McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973, 51,
1915±1919.
19. (a) Carpino, L. A.; El-Faham, A. J. Am. Chem. Soc. 1995, 117,
5401. (b) Vojkovsky, T.; Drake, B. Org. Prep. Proced. Int. 1997,
29, 497.
36. Freidinger, R. M.; Hinkle, J. S.; Perlow, D. S.; Arison, B. H.
J. Org. Chem. 1983, 48, 77±81.