2-Mercaptopyridine-1-oxide-based peptide coupling reagents

Article abstract of DOI:10.1016/S0040-4020(01)00985-1

The thiouronium salts S-(1-oxido-2-pyridinyl)-1,1,3,3-tetramethylthiouronium hexafluorophosphate (HOTT) and tetrafluoroborate (TOTT), and S-(1-oxido-2-pyridinyl)-1,3-dimethyl-1,3-trimethylenethiouronium hexafluorophosphate (HODT) and tetrafluoroborate (TODT), prepared from 2-mercaptopyridine-1-oxide and 1,1,3,3-tetramethylurea (TMU) or 1,3-dimethylpropyleneurea (DMPU), have been employed as reagents in solution and solid-phase peptide coupling chemistry. Furthermore, 2-mercaptopyridine-1-oxide has been employed as racemization-reducing additive combined with the new thiouronium salts and other frequently used peptide coupling reagents such as DCC or TBTU.

Full text of DOI:10.1016/S0040-4020(01)00985-1

TETRAHEDRON
Pergamon
Tetrahedron 57 ꢀ2001) 9607±9613
2-Mercaptopyridine-1-oxide-based peptide coupling reagents
a
Fernando Albericio, Miguel A. Bail e n, Rafael Chinchilla, David J. Dodsworth and
b
b
b
b,p,²
Carmen N a jera
a
Departamento de Qu Âõ mica Org a nica, Universidad de Barcelona, E-08028 Barcelona, Spain
Departamento de Qu Âõ mica Org a nica, Facultad de Ciencias, Universidad de Alicante, Apartado 99, 03080 Alicante, Spain
b
Received 11 June 2001; revised 7 September 2001; accepted 10 October 2001
AbstractÐThe thiouronium salts S-ꢀ1-oxido-2-pyridinyl)-1,1,3,3-tetramethylthiouronium hexa¯uorophosphate ꢀHOTT) and tetra¯uorobo-
rate ꢀTOTT), and S-ꢀ1-oxido-2-pyridinyl)-1,3-dimethyl-1,3-trimethylenethiouronium hexa¯uorophosphate ꢀHODT) and tetra¯uoroborate
ꢀ
TODT), prepared from 2-mercaptopyridine-1-oxide and 1,1,3,3-tetramethylurea ꢀTMU) or 1,3-dimethylpropyleneurea ꢀDMPU), have been
employed as reagents in solution and solid-phase peptide coupling chemistry. Furthermore, 2-mercaptopyridine-1-oxide has been employed
as racemization-reducing additive combined with the new thiouronium salts and other frequently used peptide coupling reagents such as
DCC or TBTU. q 2001 Elsevier Science Ltd. All rights reserved.
4
1
. Introduction
useful and widely used peptide coupling reagents.
Nowadays, the preferred peptide coupling reagents are
principally phosphonium ꢀ2) or aminium ꢀ3) derivatives,
where Y is a halogen or a XO group, which have become
popular because of their higher ef®ciency and lower
tendency towards racemization of the amino acid or peptide
residues. Frequently employed peptide coupling reagents
The formation of amide bonds is the main goal in the syn-
thesis of a huge array of organic compounds of biological
interest such as peptides and peptoids, and other com-
pounds containing the amide bond such as oligocarbamates,
oligoamides, b-lactams, polyenamides, benzodiazepines,
diketopiperazines, and hydantoins. The activation of carb-
oxylic acids for the formation of the amide bond is an
important process usually carried out using so-called
1
8
9
are exempli®ed by BOP ꢀ4a) and TBTU ꢀ5a), derived
from HOBt and a phosphotriamide or a urea, and more
6
b,10
6a
recently by AOP
HOAt.
ꢀ4b) and HATU ꢀ5b) derived from
2
peptide coupling reagents. The coupling reactions can be
carried out in solution or solid-phase by in situ activation of
the carboxylic acid or by the prior preparation and isolation
of an activated species. A commonly encountered problem
in peptide synthesis involves the extensive racemization of
the amino acid component, usually through oxazolone
formation, and also undesired side reactions and low
In general, both components of the coupling reagent, for
instance the XOH and the urea, should be soluble in water
to facilitate their separation from the ®nal peptide formed
especially when used in solution phase couplings. This
particular characteristic prompted us to try 2-mercapto-
pyridine-1-oxide ꢀ6) as a XSH-derived species. Compound
6 is a cheap reagent, which is commercially available as an
1
c,2,3
coupling rates especially under solid-phase conditions.
Thus, a combination of high ef®ciency during the formation
of the amide bond together with a low level of racemization
of the coupled components represent the main challenge in
the development of any new coupling reagent.
During the last few years, a plethora of coupling reagents
2
has appeared in the literature, considerably improving older
coupling methods using, for example, carbodiimides.
4
These latter reagents, in the presence of several additives
with an X±OH structure, such as 1-hydroxybenzotriazole
5
HOBt, 1a), 1-hydroxy-7-azabenzotriazole ꢀHOAt, 1b)
6
ꢀ
or hydroxylamine derivatives, such as free or polymer-
3
7
supported N-hydroxysuccinimide ꢀHOSu), are still very
Keywords: thiouronium; ureas; coupling reagents; peptides; solid-phase.
Corresponding author. Fax: 134-96-590-3549; e-mail: cnajera@ua.es
URL: www.ua.es/dept.quimorg.
p
²
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020ꢀ01)00985-1

9608
F. Albericio et al. / Tetrahedron 57 ꢀ2001) 9607±9613
Scheme 1.
aqueous solution of its sodium salt, and has been used
principally for the preparation of Barton thiohydroxamate
esters, which are precursors of radicals generated by
coupling characteristics have been compared to other
reagents including 7. The utility of the thiouronium salts 7
and 8 in solid-phase peptide synthesis ꢀSPPS) employing
hindered amino acids is also reported with particular
emphasis being placed on their ef®ciency and level of
racemization produced during the coupling reactions. We
have also studied the use of compound 6 as an additive in
combination with other peptide coupling reagents in order to
check its effectiveness as a racemization-reducing agent.
1
1
photolysis.
We have recently employed 2-mercaptopyridine-1-oxide
6) and 1,1,3,3-tetramethylurea ꢀTMU) as starting materials
for the preparation of a new class of aminium peptide
ꢀ
1
2
13
coupling reagents with a thiouronium structure. Thus,
we prepared S-ꢀ1-oxido-2-pyridinyl)-1,1,3,3-tetramethyl-
1
5
thiouronium hexa¯uorophosphate ꢀHOTT, 7a) and the
corresponding tetra¯uoroborate ꢀTOTT, 7b), and used
1
2
them for solution-phase peptide synthesis achieving yields
and levels of racemization comparable to other more expen-
sive and commonly used coupling reagents. We have also
employed these salts for the direct preparation of primary
amides by the coupling of carboxylic acids with ammonium
1
6
chloride.
2. Results and discussion
In addition, and taking into account the reported embryo-
1
7
12
The uronium salts 7 and 8
18±20
toxicity of TMU, we have also prepared the analogous
thiouronium salts 8a ꢀHODT) and 8b ꢀTODT) from a non-
toxic urea, the common organic solvent 1,3-dimethylpropyl-
can be prepared by the one-
pot phosgene-free methodology outlined in Scheme 1. The
thiouronium salts 8 were used as peptide coupling reagents
for the liquid-phase preparation of a series of di- and tri-
peptides and the results compared with those obtained with
1
8,19
eneurea ꢀDMPU). The use of compounds 8 as peptide
coupling reagents in solution has been studied and their
2
0
Table 1. Prepared peptides in solution-phase using 8 as coupling reagents
a
25
Entry
Reagent
t ꢀh)
Peptide ꢀno.)
Yield ꢀ%)
Mp ꢀ8C)
110±111
88±89
[a]
D
b
1
2
3
4
5
6
7
8
9
8a
8b
8a
8b
8b
8a
8b
8b
8a
8b
8b
8a
8b
8a
8b
8a
8b
4
4
4
4
4
Cbz-Val-Val-OMe ꢀ13a)
Cbz-Val-Aib-OMe ꢀ13b)
82
83
60
76
66
66
70
222.6b
222.3
220.0
c
c
220.0
216.0
Boc-Val-Aib-OMe ꢀ13c)
Boc-Aib-Val-OMe ꢀ13d)
148±149
110±111
4
4
24
24
24
24
4
4
4
4
4
Cbz-Aib-Aib-OMe ꢀ13e)
Boc-Aib-Aib-OMe ꢀ13f)
74 ꢀ68)
66
83
109±110
86±87
10
11
12
13
14
15
16
17
d
d
Fmoc-Aib-Aib-OMe ꢀ13g)
Bz-Leu-Gly-OEt ꢀ13h)
47 ꢀ60 )
127±128
151±152
e
64
70
220.5
221.0
e
f
Cbz-Gly-Phe-Val-OMe ꢀ13i)
g
63
84
h
d
i
Cbz-MeVal-Val-OMe ꢀ13j)
81
75
271.5i
273.2
d
4
a
b
c
d
e
f
Isolated pure compounds after aqueous work-up. Based in the starting amino acid. In parentheses, yields using 7b as coupling reagent.
25a
Lit. [a]
20
D
ˆ228 ꢀc 1, EtOH).
25b
Lit. [a]
D
ˆ224 ꢀc 1, EtOH).
After column chromatography ꢀsilica gel, hexane/AcOEt).
ˆ234 ꢀc 3.1, EtOH).
23
Lit. [a]
20
D
Underlined the new formed bond.
Diastereomeric ratio ꢀ H NMR): 10:0.8.
g
h
i
1
1
Diastereomeric ratio ꢀ H NMR): 10:1.
22b
Lit. [a]
20
D
ˆ290 ꢀc 1, EtOH).

F. Albericio et al. / Tetrahedron 57 ꢀ2001) 9607±9613
9609
Table 2. Use of 6 as additive in Anteunis' test ꢀsynthesis of 13i)
good isolated yields of the corresponding peptide 13j when
coupled with Val-OMe using 8 ꢀTable 1, entries 16 and 17)
values comparable to those obtained using 7 ꢀ80% for 7a
a
b
Entry
Reagent16 ꢀ1 equiv.)
Yield ꢀ%)
d,l ꢀ%)
1
2
c
1
2
3
4
5
6
7
8
9
DCC16
DCC
81
6.5
and 82% for 7b).
d
36
TBTU ꢀ5a)16
TBTU ꢀ5a)
HOTT ꢀ7a)16
HOTT ꢀ7a)
TOTT ꢀ7b)16
TOTT ꢀ7b)
HODT ꢀ8a)16
HODT ꢀ8a)
TODT ꢀ8b)16
TODT ꢀ8b)
90
1.4
1.4
The extent of racemization under solution-phase conditions
using these reagents was examined by performing Young's
d
87
68
83
71
72
63
85
84
3.8
9.0
2.4
9.0
3.8
7.4
2.9
9.0
2
3
test ꢀthe coupling of Bz-Leu-OH and Gly-OEt´HCl, Table
2
4
1, entries 12 and 13) and Anteunis' test ꢀthe coupling of
Cbz-Gly-Phe-OH and Val-OMe´HCl, Table 1, entries 14
and 15). A higher level of racemization was observed in
Young's test using DMPU-derived reagents 8 when
10
11
12
1
2
compared to the corresponding TMU derivatives 7.
Thus, for reagents 8, the level of racemization was ca.
9%, a value close to that obtained when using HATU
a
Isolated pure compound after aqueous work-up. Based on the starting
amino acids.
Measured by H NMR.
1
12
b
c
d
1
ꢀ5b), whereas the corresponding value was only 3.7%
1
2
using reagent 7b. On the other hand, when Anteunis'
test was performed, the DMPU-derived salt 8a gave the
lowest level of epimerization [diastereomeric ratio ꢀ H
The reactions was performed in CH
Measured by HPLC ꢀRef. 9).
2 2
Cl .
1
NMR)ˆ10:0.8] in the corresponding tripeptide 13i ꢀTable
7
. In all cases we checked the ef®ciency of these reagents
1, entry 14), reagents 7 giving slightly higher diastereomer
ratios ꢀ10:1, NMR).
1
2
performing rather dif®cult couplings using hindered amino
acids such as Val or, specially, a,a-dialkyl amino acids such
as a-aminoisobutyric acid ꢀAib) or N-methylated amino
acids. The Aib±Aib coupling is described as being particu-
larly dif®cult for both solution and solid-phase strategies,
whereas N-methylated amino acids usually prove resistant
in coupling reactions.
The reactivity of peptide coupling reagents can be enhanced
in some cases by the addition of various additives which at
the same time also serve to reduce the extent of racemi-
2
1
1
zation. For instance, a serious drawback to the use of carbo-
2
2a
diimides as peptide coupling reagents is the high level of
racemization usually observed in the coupled product.
However, in the presence of additives with a XOH structure,
usually a hydroxylamine derivative, yields are increased and
Thus, different Boc-, Cbz-, Fmoc- and Bz-N-protected
amino acids or dipeptides were reacted with ethyl or methyl
ester amino acid hydrochlorides in the presence of the
uronium salts 8 and triethylamine as base using DMF as
solvent. After work-up, the corresponding pure ꢀ H NMR,
3
4
,7
racemization levels are lowered. Moreover, it has been
shown that racemization is decreased during the coupling
of peptide fragments in solution when HOBt ꢀ1a) or HOAt
1
2
6
00 MHz) crude peptides 13 were isolated with the yields
ꢀ1b) or are added to aminium-derived coupling reagents.
shown in Table 1. The reaction time was always 4 h, except
when the dif®cult Aib±Aib coupling was performed, which
required 24 h for completion. A certain tendency was
observed for the hexa¯uorophosphate 8b to afford higher
yields than the corresponding tetra¯uoroborate 8a, a feature
These antecedents prompted us to explore the use of
2-mercaptopyridine-1-oxide ꢀ6) as an additive when
combined with other reagents commonly used for peptide
couplings reagents, and also with the thiouronium salts 7
and 8. Thus, the Anteunis' tripeptide 13i was obtained by
using a mixture of 6 ꢀ1 equiv.) and a carbodiimide such as
N,N-dicyclohexylcarbodiimide ꢀDCC), affording a product
with a dramatically lowered racemization level when
compared to the values reported in the absence of any
1
2
already observed with reagents 7. In general, the DMPU
derivatives 8 afforded similar or even higher yields than
1
2
the corresponding TMU derivatives 7.
N-Boc-protected amino acids are particularly prone to
N-carboxyanhydride formation,
Moreover,
2
2b
9
and it is remarkable
additive ꢀcompare Table 2, entries 1 and 2), although not
as much as the reported lowering when HOBt was used,
that the yield in the preparation of the `dif®cult' dipeptide
Boc-Aib-Aib-OMe was 83% when the DMPU-derivative 8b
was employed ꢀTable 1, entry 10), but only 70% when
TBTU ꢀ5a) was used for comparison. Furthermore, a
N-methylated amino acid, such as Cbz-MeVal-OH afforded
2
4
which gave no epimerization at all. However, when 6
was employed as additive in combination with TBTU
ꢀ5a), no lowering of the racemization degree was observed
9
when compared to the value measured without additive
Table 3. Ef®ciency of the reagents 7 and 8 in SPPS
Entry
Reagent
Peptides
H-Tyr-Aib-Aib-Phe-Leu-NH
1
2
,
H-Tyr-Aib-Phe-Leu-NH
2
,
a
a
4 ꢀ%)
15 ꢀ%)
1
2
3
4
5
TBTU ꢀ5a)
HOTT ꢀ7a)
TOTT ꢀ7b)
HODT ꢀ8a)
TODT ꢀ8b)
29
24
19
10
14
71
76
81
90
86
a
Measured by integration of the signals in the HPLC at 220 nm ꢀsee Section 3).

9610
F. Albericio et al. / Tetrahedron 57 ꢀ2001) 9607±9613
Table 4. Racemization studies in SPPS using 7 and 8 with Ser and His models
Entry
Reagent
Peptides
a
a
Gly-ꢀl)-Ser-Phe-NH , 16 ꢀ%)
2
2
Gly-ꢀl)-His-Phe-NH , 17 ꢀ%)
1
2
3
4
5
TBTU ꢀ5a)
HOTT ꢀ7a)
TOTT ꢀ7b)
HODT ꢀ8a)
TODT ꢀ8b)
.99
.99
.99
.99
.99
97.1
98.4
98.6
98.1
98.0
a
Measured by integration of the signals in the HPLC at 220 nm ꢀsee Section 3).
ꢀ
compare Table 2, entries 3 and 4). A signi®cant effect was
when the thiopyridone derivatives 7 were used ꢀTable 4,
entries 2 and 3).
observed when an extra equivalent of 6 was used with
reagents 7 or 8, affording in all cases a decrease in
racemized product and an increase in the ®nal yield of the
tripeptide ꢀTable 2, entries 5±12).
In conclusion, 2-mercaptopyridine-1-oxide-derived thio-
uronium salts from TMU [HOTT ꢀ7a) and TOTT ꢀ7b)] or
DMPU [HODT ꢀ8a) and TODT ꢀ8b)] can be prepared
following an easily scalable one pot procedure from
economical reagents and can be employed as ef®cient
peptide coupling reagents. The coupling of hindered
amino acids in solution using these reagents affords
generally higher yields for the DMPU derivatives 8 when
compared to the TMU derivatives 7. However, the results
obtained in SPPS reveal a higher ef®ciency for the TMU
derivatives 7, together with lower racemization levels. This
could be explained by the fact that, in general, DMPU
derivatives are less stable than the corresponding TMU
In order to check the ef®ciency of 7 and 8 in SPPS, we
performed the manual stepwise solid-phase assembly of a
typical model pentapeptide analog of Leu-enkephalinamide,
6
b,27a
H-Tyr-Aib-Aib-Phe-Leu-NH2 ꢀ14),
which contains
Aib-OH residues at positions 2 and 3 instead of Gly-OH,
following a Fmoc/tBu protection scheme. A polyꢀethylene-
glycol)±polystyrene ꢀPEG±PS)-resin bearing a peptide
amide linker derived from p-{a-[1-ꢀ9H-¯uoren-9-yl)meth-
oxyformamido]-2,4-dimethoxybenzyl}phenoxyacetic acid
ꢀ
Rink) was used as a solid support, in DMF as solvent and
using diisopropylethylamine ꢀDIEA) as base ꢀsee 3). TBTU
5a) ꢀ®ve-fold excess, single coupling) was employed as
coupling reagent for normal amino acids, whereas 7 or 8
®ve-fold excess, single coupling) were used for the
3
derivatives and in solid-phase the reaction usually has
ꢀ
lower kinetics. The addition of an extra amount of
2-mercaptopyridine-1-oxide to the thiouronium salts 7 and
8 improves the ®nal yields and lowers lowering the extent of
racemization. The results obtained show that the reagents 7
and 8 are comparable to other peptide coupling reagents
presently available.
ꢀ
coupling of the dif®cult Aib-Aib sequence. Table 3 shows
the amount of pentapeptide ꢀ14) and des-Aib tetrapeptide
ꢀ
15) obtained after cleavage, together with the result for
Aib±Aib coupling using TBTU ꢀ5a) for comparison
Table 3, entry 1). In all cases the ef®ciency in the Aib±
ꢀ
Aib coupling was lower than with TBTU, although the
result was not very different when using the TMU-derived
tetra¯uoroborate 7a ꢀTable 3, entry 2). The lowest ef®ciency
was observed with the DMPU-derived thiouronium 8a
3. Experimental
3.1. General
ꢀ
Table 3, entry 4).
All reactions carried out during the preparation of the
thiouronium salts were moisture-protected using a drying
tube with calcium chloride. 2-Mercaptopyridine-1-oxide
The extent of racemization induced by these reagents in
SPPS was studied performing the manual stepwise solid-
phase assembly of two model tripeptides containing Ser
ꢀ
1
The synthesis was performed following the same methodol-
ogy as above, using TBTU ꢀ5a) except when coupling the
Ser or His residues to Phe where 7 or 8 were used. In both
cases, an all-TBTU ꢀ5a) synthesis was including for
comparison ꢀTable 4, entry 1). The extent of racemization
was obtained by measuring the ratio between diastereomeric
peptides containing l- and d-Ser, and l- and d-His ꢀHPLC),
the yield of the l-containing peptides being presented
2
8
ꢀ6) was obtained according to the literature procedure.
Reagents and solvents from commercial suppliers were
2
7b
21
used as provided. PEG±PS resin ꢀ0.19 mmol g ) and the
H-Gly-Ser-Phe-NH , 16) and His ꢀH-Gly-His-Phe-NH ,
2
7) both amino acids especially prone to racemization.
2
7c,d
2
Fmoc-Rink handle were obtained from the BioSearch
division of PerSeptive Biosystems ꢀFramingham, MA).
Melting points are uncorrected. IR spectra were recorded
with a Nicolet 510 P-FT and only the structurally important
peaks are indicated. NMR spectra were measured with a
1
13
Bruker AC-300 at 300 MHz for H and 75 MHz for
C
using TMS as internal standard. Optical rotations were
measured with a Jasco DIP-1000 polarimeter. MALDI-
TOF spectra were recorded with a Perspective Biosystems
Voyager DEe-RP spectrometer using a 2,5-dihydroxy-
benzoic acid matrix. Analytical TLC was performed with
Schleicher and Schuell F1400/LS silica gel plates and the
spots were visualized with UV light at 254 nm or by
charring with 0.3% ninhydrin. Flash chromatography was
carried out with Merck silica gel 60 ꢀ0.040±0.063 nm).
ꢀ
Table 4). The extent of racemization in the Ser-containing
model tripeptide ꢀ16) was very low in all cases and similar
to that observed using TBTU ꢀ5a) ꢀTable 4). However, for
the more sensitive His-containing tripeptide ꢀ17), the
racemization when using reagents 7 and 8 was lower in all
cases when compared to TBTU ꢀ5a) ꢀTable 4), especially

F. Albericio et al. / Tetrahedron 57 ꢀ2001) 9607±9613
9611
Reverse-phase HPLC analyses were carried out on Nucleo-
sil C18 columns ꢀ25£0.5 cm, 10 mm) using linear gradients
of water/0.045% TFA ꢀsolvent A) and MeCN/0.036% TFA
Anal. Calcd for C H N OSBF : C, 40.60; H, 4.96; N,
11 16 3 4
12.92. Found: C 40.52; H, 4.32; N, 12.60.
2
1
ꢀ
solvent B) ꢀ¯ow: 1 mL min , detection wavelength:
20 nm). Leu-enkephalinamide derivative 14 was analysed
3.3. General procedure for the peptide coupling
reactions in solution-phase
2
using a linear gradient from 25 to 30% of B in 30 min
followed by 100% of B for 5 min. The Ser and His racemi-
zation models 16 and 17 were analysed using a linear
gradient from 0 to 20% of B in 20 min, followed by a
gradient from 20 to 100% of B in 20 min and 100% of B
for 5 min. The HPLC signals were measured at 220 nm.
A solution of the N-protected amino acid ꢀ1 mmol), the
amino acid ester hydrochloride ꢀ1 mmol), triethylamine
ꢀ0.28 mL, 2 mmol) and the coupling reagent 8 ꢀ1 mmol)
in DMF ꢀ5 mL) was stirred at room temperature until dis-
appearance of the free esteri®ed amino acid ꢀTLC, nin-
hydrin). Saturated NaCl ꢀ25 mL) was added, and the
mixture was extracted with AcOEt ꢀ3£25 mL). The organic
layers were washed with 2N HCl ꢀ2£10 mL), saturated
3
.2. Synthesis of thiouronium salts 7 and 8
NaHCO ꢀ2£10 mL) and water ꢀ6£10 mL). The organics
3
S-ꢀ1-Oxido-2-pyridinyl)-1,1,3,3-tetramethylthiouronium
hexa¯uorophosphate ꢀHOTT, 7a) and the corresponding
were dried ꢀNa SO ), ®ltered and evaporated ꢀ15 Torr)
2
4
affording the corresponding peptides 13 ꢀsee Table 1).
1
2
tetra¯uoroborate ꢀTOTT, 7b) were prepared as reported.
S-ꢀ1-Oxido-2-pyridinyl)-1,3-dimethylpropylenethiouronium
hexa¯uorophosphate ꢀHODT, 8a) and the corresponding
tetra¯uoroborate ꢀTODT, 8b) were prepared as follows: to
a solution of 1,3-dimethylpropyleneurea ꢀDMPU) ꢀ4.8 mL,
25a 1
3
0
2
.3.1. Cbz-Val-Val-OMe (13a).
.87±0.98 ꢀm, 12H, 2ꢀCH ) CH), 2.04±2.19 ꢀm, 2H,
H NMR ꢀCDCl ) d
3
3
2
CHꢀCH ) ), 3.73 ꢀs, 3H, CH O), 4±4.10 ꢀm, 1H,
3
3
2
CHCONH), 4.55 ꢀdd, Jˆ8.5, 4.9 Hz, 1H, CHCO Me),
2
4
0 mmol) and DMF ꢀ0.3 mL) in CH Cl ꢀ40 mL) was added
2 2
5
.12 ꢀs, 2H, CH ), 5.41 ꢀd, Jˆ8.5 Hz, 1H, NH), 6.44 ꢀd,
2
dropwise oxalyl chloride ꢀ4.2 mL, 48 mmol) at room
temperature. The solution was stirred for 1 h at room
temperature and then re¯uxed for 24 h. The solvent was
evaporated ꢀ15 Torr) and the resulting solid was stirred
with portions of CH Cl ꢀ2£10 mL) followed by evapora-
Jˆ8 Hz, 1H, NH), 7.34 ꢀm, 5H, ArH).
25b
1
3
0
ꢀ
3
.3.2. Cbz-Val-Aib-OMe (13b).
H NMR ꢀCDCl ) d
3
.94 ꢀ2d, Jˆ6.7 Hz, 6H, ꢀCH ) CH), 1.51 ꢀs ancho, 6H,
3
2
2
2
CH ) C), 2.10 ꢀm, 1H, CHꢀCH ) ), 3.70 ꢀs, 3H, CH O),
3
2
3 2
3
tion of the organics ꢀ15 Torr) after each treatment. The
crude chlorouronium salt obtained was dissolved in
MeCN ꢀ40 mL) and KPF6 ꢀ8.8 g, 48 mmol) or NaBF4
.96 ꢀdd, Jˆ14, 7 Hz, 1H, CHCO); 5.10 ꢀs, 2H, CH ),
2
5
.56, 6.75 ꢀ2 broad s, 2H, 2NH), 7.29±7.35 ꢀm, 5H, ArH).
ꢀ
5.27 g, 48 mmol) were added. The mixture was stirred at
29 1
.3.3. Boc-Val-Aib-OMe (13c). H NMR ꢀCDCl ) d 0.85
3
ꢀ
ꢀ
4
3
room temperature for 24 h and to the resulting suspension
was added N-hydroxy-2-pyridinethione ꢀ6) ꢀ5.1 g, 40
mmol). Triethylamine ꢀ6.7 mL, 48 mmol) was added drop-
wise keeping the temperature below 258C and the resulting
suspension was stirred at room temperature for 4 h and at
2d, Jˆ7 Hz, 6H, ꢀCH ) CH), 1.37±1.45 ꢀ3s, 15H, ꢀCH ) C,
3
2
3 3
CH ) C), 2.10 ꢀm, 1H, CHꢀCH ) ), 3.65 ꢀs, 3H, CH O),
3 2 3 2 3
.45 ꢀdd, Jˆ8.5, 4.9 Hz, 1H, CHCO), 4.97, 6.96 ꢀ2 broad
s, 2H, 2NH).
2
9 1
.3.4. Boc-Aib-Val-OMe (13d). H NMR ꢀCDCl ) d 0.90
5
08C for 1 h. The suspension was ®ltered through a plug of
3
3
celite, the solvents were evaporated ꢀ15 Torr) and the
thiouronium salt 8a was obtained after crystallization with
MeOH/isopropanol ꢀ12.4 g, 81%). Compound 8b was
isolated after stirring in EtOH at 408C for 1 h, cooling at
ꢀ
2d, Jˆ7 Hz, 6H, ꢀCH ) CH), 1.44±1.52 ꢀ3s, 15H, ꢀCH ) C,
3
2
3 3
ꢀCH ) C), 2.16 ꢀm, 1H, CHꢀCH ) ), 3.72 ꢀs, 3H, CH O),
3 2 3 2 3
4
.52 ꢀdd, Jˆ8.5, 4.9 Hz, 1H, CHCO), 4.93, 7.02 ꢀ2 broad
s, 2H, 2NH).
4
8C and ®ltration ꢀ7.8 g, 60%).
2
5b 1
3
.3.5. Cbz-Aib-Aib-OMe (13e).
H NMR ꢀCDCl ) d
3
3.2.1. S-(1-Oxido-2-pyridinyl)-1,3-dimethylpropylene-
uronium hexa¯uorophosphate (HODT, 8a). Mp 111±
1
.50, 1.51 ꢀ2s, 12H, 2ꢀCH ) C), 3.71 ꢀs, 3H, CH O), 5.10
3
2
3
ꢀs, 2H, CH ), 5.34, 6.90 ꢀ2 broad s, 2H, 2NH), 7.30±7.40 ꢀm,
5H, ArH).
2
2
21
1
1
NMR ꢀCD CN) d 2.21±2.23 ꢀm, 2H, CH CH N), 3.37 ꢀs,
128C. IR ꢀKBr) n 1629 ꢀCvN), 844 ꢀPF ) cm . H
6
3
2
2
2
5b 1
6
H, CH ), 3.65 ꢀt, Jˆ6.1 Hz, 4H, CH N), 7.35±7.47 ꢀm, 3H,
3
2
1
3.3.6. Boc-Aib-Aib-OMe (13f).
ꢀs, 9H, ꢀCH ) C), 1.47, 1.54 ꢀ2s, 12H, 2ꢀCH ) C), 3.72 ꢀs,
3 3 3 2
3H, CH O), 5.10, 7.10 ꢀ2 broad s, 2H, 2NH).
3
H NMR ꢀCDCl ) d 1.45
3
3
ArH), 8.30 ꢀd,1H, Jˆ5.5 Hz, ArH). C NMR ꢀCD CN) d
3
2
0.4 ꢀ1C, CH ), 44.7 ꢀ2C, CH ), 51.6 ꢀ2C, CH ), 125.4,
2 3 2
1
Anal. Calcd for C H N OSPF : C, 34.47; H, 4.21; N,
26.2, 128.8, 140.2, 144.4 ꢀ5C, ArC), 160.4 ꢀ1C, CvN).
29 1
3.3.7. Fmoc-Aib-Aib-OMe (13g). H NMR ꢀCDCl ) d
1
1
16
3
6
3
1
0.96. Found: C, 34.28; H, 4.29; N, 11.02.
1.50, 1.52 ꢀ2s, 12H, 2ꢀCH ) C), 3.70 ꢀs, 3H, CH O), 4.19
3
2
3
ꢀ
t, Jˆ6.7 Hz, 1H, CH), 4.39 ꢀd, Jˆ6.7 Hz, 2H, CH CH),
2
3
.2.2. S-(1-Oxido-2-pyridinyl)-1,3-dimethylpropylene-
uronium tetra¯uoroborate (TODT, 8b). Mp 121±1228C.
5.55, 6.90 ꢀ2 broad s, 2H, 2NH), 7.26±7.75 ꢀm, 8H, ArH).
2
21
IR ꢀKBr) n 1620 ꢀCvN), 1025 ꢀBF ) cm . H NMR
1
23 1
3.3.8. Bz-Leu-Gly-OEt (13h). H NMR ꢀCDCl ) d 0.98
4
3
ꢀ
CD CN) d 2.20±2.23 ꢀm, 2H, CH CH N), 3.37 ꢀs, 6H,
3
ꢀd, Jˆ6.1 Hz, 6H, ꢀCH ) CH), 1.26 ꢀt, Jˆ7.3 Hz, 3H,
3 2
2
2
CH ), 3.65 ꢀt, Jˆ6.1 Hz, 4H, CH N), 7.34±7.47 ꢀm, 3H,
CH CH ), 1.65±1.84 ꢀm, 3H, CH CH, ꢀCH )CH)), 4±4.10
2
3
2
3
2
3
1
3
ArH), 8.30 ꢀd, Jˆ5.5 Hz, 1H, ArH). C NMR ꢀCD CN) d
ꢀm, 2H, CH NH), 4.16±4.24 ꢀq, 2H, Jˆ7.3 Hz, CH CH ),
3
2
2
3
2
1
0.4 ꢀ1C, CH ), 44.8 ꢀ2C, CH ), 51.7 ꢀ2C, CH ), 125.4,
3
4.74 ꢀm, 1H, CHNH), 6.60, 6.71 ꢀ2 broad s, 2NH), 7.41±
7.80 ꢀm, 5H, ArH).
2
2
26.2, 128.8, 140.2, 144.4 ꢀ5C, ArC), 160.4 ꢀ1C, CN).

9612
F. Albericio et al. / Tetrahedron 57 ꢀ2001) 9607±9613
2
.3.9. Cbz-Gly-Phe-Val-OMe (13i). l-Phe-containing
4
3
DMF ꢀ5£2 mL) and CH Cl ꢀ3£2 mL). The formed resin
2
2
1
epimer: H NMR ꢀCDCl ) d 0.75 ꢀ2d, Jˆ7 Hz, 6H,
was washed with DMF ꢀ5£2 mL) and treated with a mixture
of Fmoc-TyrꢀtBu)-OH ꢀ22 mg, 5 equiv.), TBTU ꢀ15 mg,
5 equiv.) and DIEA ꢀ8 mL, 10 equiv.) in DMF ꢀ0.5 mL).
After stirring at 258C for 2 h, the Fmoc group was depro-
tected with 20% piperidine in DMF ꢀ2 mL, 1 min12 mL,
15 min), followed by washes with DMF ꢀ5£2 mL) and
CH Cl ꢀ3£2 mL). Final cleavage was achieved after
3
ꢀ
CH ) CH), 1.96 ꢀm, 1H, CHꢀCH ) ), 2.94 ꢀm, 2H,
3 2 3 2
CH Ph), 3.57 ꢀs, 3H, CH O), 3.77 ꢀd, Jˆ5.5 Hz, 2H,
2
3
HNCH CO), 4.34 ꢀdd, Jˆ8, 5.5 Hz, 1H, HNCH-iPr), 4.75
2
ꢀ
m, 1H, CH-Bn), 5.01 ꢀs, 2H, CH OCONH), 5.75 ꢀt, Jˆ
2
5
7
.5 Hz, 1H, NHCH ), 6.86 ꢀd, Jˆ8 Hz, 1H, NHCH-iPr),
2
.04±7.26 ꢀm, 10H, 9ArH, NH).
2
2
swelling the obtained Tyr-Aib-Aib-Phe-Leu-Rink-PEG±
2
2b 1
3
0
2
4
.3.10. Cbz-MeVal-Val-OMe (13j).
.76±0.96 ꢀm, 12H, 2ꢀCH ) CH), 2.10, 2.30 ꢀ2m, 2H,
H NMR ꢀCDCl ) d
3
PS resin with CH Cl ꢀ3£2 mL) and treatment with TFA/
2
2
3
2
triethylsilane ꢀTES)/H O ꢀ95:3:2, 0.5 mL) with stirring for
2
CHꢀCH ) ), 2.90 ꢀs, 3H, CH N), 3.71 ꢀs, 3H, CH O),
2
2 h at 258C. The suspension was ®ltered and the solvent was
removed under a stream of nitrogen. The resulting residue
was triturated with terc-butyl methyl ether and cooled in a
freezer overnight. The mixture was centrifuged ꢀ4000 rpm)
for 10 min and the solvent was decanted. The resulting solid
was dried under a stream of nitrogen and liophilized giving
a mixture of peptides 22 and 23 which was analyzed by
HPLC ꢀsee Table 3).
3
3
3
.10 ꢀm, 1H, CHNMe), 4.48 ꢀdd, Jˆ8.8, 5.2 Hz,
CHNHCO), 5.17 ꢀAB, Jˆ11.6, 12.8 Hz, 2H, CH ), 6.55
2
ꢀ
d, Jˆ8.8 Hz, 1H, NH), 7.36 ꢀm, 5H, ArH).
3.4. General procedure for the peptide coupling
reactions using 6 as additive
A solution of the N-protected amino acid ꢀ1 mmol), the
amino acid ester hydrochloride ꢀ1 mmol), triethylamine
3.5.1. Tyr-Aib-Aib-Phe-Leu-NH (14). HPLC: t ˆ16 min.
MALDI-TOF MS calcd for C H N O : 610.7. Found:
32 46 6 6
611.3 ꢀM 1H).
2
R
ꢀ
0.28 mL, 2 mmol), the coupling reagent ꢀ1 mmol) and
-mercaptopyridine-1-oxide ꢀ6) ꢀ127 mg, 1 mmol) in
1
2
DMF ꢀ5 mL) was stirred at room temperature for 4 h.
Saturated NaCl ꢀ25 mL) was added, and the mixture was
extracted with AcOEt ꢀ3£25 mL). The organic layers were
3.5.2. Tyr-Aib-Phe-Leu-NH (15). MALDI-TOF MS calcd
2
1
for C H N O : 525.6. Found: 526.5 ꢀM 1H). HPLC:
2
8
39
5
5
washed with 2N HCl ꢀ2£10 mL), saturated NaHCO
tRˆ17 min.
3
ꢀ
ꢀ
2£10 mL) and water ꢀ6£10 mL). The organics were dried
Na SO ), ®ltered and evaporated ꢀ15 Torr) affording the
2
4
peptide 13i ꢀsee Table 2).
3.6. SPPS of tripeptides 16 and 17
3
.5. SPPS of Leu-enkephalinamide derivative 14
To a Rink PEG±PS resin prepared as in Section 3.5. ꢀ1 g),
was added a solution of Fmoc-Phe-OH ꢀ0.37 g, 5 equiv.),
TBTU ꢀ0.31 g, 5 equiv.) and DIEA ꢀ0.323 mL, 10 equiv.) in
DMF ꢀ1 mL). The mixture was stirred for 2 h and the resin
2
1
To a neutralized PEG±PS resin ꢀ1 g, 0.19 mmol g ) was
added the preformed Fmoc-Rink handle ꢀ0.2 g, 0.38 mmol),
TBTU ꢀ0.31 g, 5 equiv.), HOAt ꢀ0.13 g, 5 equiv.), and
DIEA ꢀ0.323 mL, 10 equiv.) dissolved in DMF ꢀ2 mL).
After overnight coupling, the resulting Fmoc-Rink±PEG±
PS resin was washed with DMF ꢀ5£10 mL) and the Fmoc
group was removed with 20% piperidine in DMF ꢀ5 mL,
was washed with DMF ꢀ5£10 mL) and CH
Cl
ꢀ3£10 mL).
2
2
A qualitative ninhydrin test was used to determine com-
pletion of all couplings. The Fmoc group was removed
with 20% piperidine in DMF ꢀ10 mL, 1 min110 mL,
15 min), followed by washes with DMF ꢀ5£10 mL) and
1
min15 mL, 15 min), followed by washes with DMF
CH
2
Cl
2
ꢀ3£10 mL). Portions of this Phe-Rink±PEG±PS
ꢀ
5£10 mL), CH Cl ꢀ5£10 mL) and MeOH ꢀ3£10 mL)
resin ꢀ50 mg) were treated with a solution of DIEA ꢀ8 mL,
10 equiv.), the coupling reagent 7, 8 or TBTU ꢀ5 equiv.) and
Fmoc-SerꢀtBu)-OH ꢀ18 mg, 5 equiv., for the synthesis of
16) or Fmoc-HisꢀTrt)-OH ꢀ29 mg, 5 equiv., for the synthesis
of 17) in DMF ꢀ0.5 mL), and the mixture was stirred at 258C
for 2 h. The resin was washed with DMF ꢀ5£10 mL) and
2
2
and drying in vacuo at 258C. The obtained resin ꢀ1 g) was
pre-conditioned with DMF ꢀ5£10 mL) and a solution of
Fmoc-Leu-OH ꢀ0.34 g, 5 equiv.), TBTU ꢀ0.31 g, 5 equiv.)
and DIEA ꢀ0.323 mL, 10 equiv.) in DMF ꢀ1 mL) was added
stirring the mixture for 2 h and washing the resin with DMF
ꢀ
5£10 mL) and CH Cl ꢀ3£10 mL). The Fmoc group was
CH
Cl
2
2
ꢀ3£10 mL) and the Fmoc was removed with 20%
2
2
removed with 20% piperidine in DMF ꢀ5 mL, 1 min15 mL,
5 min), followed by washes with DMF ꢀ5£5 mL) and
piperidine in DMF ꢀ2 mL, 1 min12 mL, 15 min), followed
Cl
1
by washes with DMF ꢀ5£10 mL) and CH
2
2
ꢀ3£10 mL).
CH Cl ꢀ3£5 mL). Following the same procedure, the Phe
The resulting resin was treated with a mixture of Fmoc-
Gly-OH ꢀ14 mg, 5 equiv.), TBTU ꢀ15 mg, 5 equiv.) and
DIEA ꢀꢀ8 mL, 10 equiv.) in DMF ꢀ0.5 mL). After stirring
at 258C for 2 h, the Fmoc group was deprotected with
20% piperidine in DMF ꢀ2 mL, 1 min12 mL, 15 min),
2
2
and ®rst Aib residues were introduced using Fmoc-Phe-OH
and Fmoc-Aib-OH. Qualitative ninhydrin test was used to
determine completion of all couplings. The obtained Aib-
Phe-Leu-Rink±PEG±PS resin was washed with MeOH
ꢀ
5
ꢀ
3£5 mL) and dried in vacuo at 258C. To a portion of
0 mg of this resin was added a mixture of Fmoc-Aib-OH
15 mg, 5 equiv.), DIEA ꢀ8 mL, 10 equiv.) and the coupling
followed by washes with DMF ꢀ5£2 mL) and CH
Cl
2 2
ꢀ3£2 mL). Final cleavage and treatment as in Section 3.5
afforded peptides 16 or 17 which were analyzed by
HPLC.
reagents 7, 8, or TBTU ꢀ5 equiv.) in DMF ꢀ0.5 mL). The
mixture was stirred at 258C for 2 h and the resin was washed
with DMF ꢀ5£2 mL) and CH Cl ꢀ3£2 mL). Deprotection
3.6.1. Gly-Ser-Phe-NH
(16). HPLC: t
l-Ser). MALDI-TOF MS calcd for C14
ˆ14.7 min ꢀwith
: 308.4.
2
2
2
R
of the Fmoc was performed with 20% piperidine in DMF
H
20
N
4
O
4
1
Found: 308.6 ꢀM ).
ꢀ
2 mL, 1 min12 mL, 15 min), followed by washes with

F. Albericio et al. / Tetrahedron 57 ꢀ2001) 9607±9613
9613
3
l-His), 17 min ꢀwith d-His). MALDI-TOF MS calcd for
.6.2. Gly-His-Phe-NH (17). HPLC t ˆ15.5 min ꢀwith
1
C H N O : 358.3. Found: 358.5 ꢀM ).
S-ꢀazabenzotriazol-yl)-1,1,3,3-bisꢀtetramethylene)thiouronium
2
R
6
hexa¯uorophosphate ꢀHAPyTU), S-ꢀ7-azabenzotriazol-yl)-
d
14
1,1,3,3-tetramethylthiouronium hexa¯uorophosphate ꢀHATTU)
and bisꢀtetramethylene)penta¯uorothiophenoxyformamidinium
1
7
22
6
3
6
d
hexa¯uorophosphate ꢀHpySPfp).
Acknowledgements
14. Klose, J.; Henklein, P.; El-Faham, A.; Carpino, L. A.; Bienert,
M. In Peptides 1998. Proceedings of the 25th European
Peptide Symposium; Bajusz, S., Hudecz, F., Eds.; Akad e miai
Kiad o : Budapest, 1999; p 204.
We thank the Direcci o n General de Ense nÄ anza Superior
e Investigaci o n Cient Âõ ®ca ꢀprojects 1FD97-0721 and
BQU2000-0235) of the Ministerio de Educaci o n y Cultura
MEC) and ASAC Pharmaceuticals International for
nancial support.
15. HOTT has also been used for the preparation of Barton esters,
useful in photolysis reactions: ꢀa) Garner, P.; Anderson, J. T.;
Dey, S.; Youngs, W. J.; Galat, K. J. Org. Chem. 1998, 63,
ꢀ
®
5
732±5733. ꢀb) Takasu, K.; Mizutani, S.; Noguchi, M.;
Makita, K.; Ihara, M. Org. Lett. 1999, 1, 391±393. ꢀc) Garner,
P.; Anderson, T. Org. Lett. 1999, 1, 1057±1059.
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28. N-Hydroxy-2-pyridinethione ꢀ6) is commercially available or
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3. Some thiouronium salts have recently been reported, such as