The retro-mannich cleavage of δ1,δ1'-tryptophan dimers

Article abstract of DOI:10.1016/S0968-0896(98)00071-6

Under acidic conditions tryptophan sidechains crosslink to form δ₁, δ₁'-tryptophan dimers through a Mannich-type mechanism. Tryptophan dimers are readily cleaved at high temperatures under acidic conditions making it impossible to isolate tryptophan dimers under standard conditions of acidic protein hydrolysis. In a prescriptive sense this cleavage can be used to recover peptides that have undergone tryptophan crosslinking, although the yields drop with increasing peptide length due to competitive cleavage of the amide bonds. The best conditions for cleavage involve heating the dimeric peptides in dilute ethanolic HCl at 150°C in the presence of ten equivalents of ethanedithiol. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00071-6

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 975±981
0
The Retro-Mannich Cleavage of ꢀ ,ꢀ -Tryptophan Dimers
1
1
Bluegrass Biggs, Alice L. Presley and David L. Van Vranken*
Department of Chemistry, University of California, Irvine, CA 92697, USA
Received 19 November 1997; accepted 3 February 1998
0
AbstractÐUnder acidic conditions tryptophan sidechains crosslink to form d
1
, d
1
-tryptophan dimers through a Man-
nich-type mechanism. Tryptophan dimers are readily cleaved at high temperatures under acidic conditions making it
impossible to isolate tryptophan dimers under standard conditions of acidic protein hydrolysis. In a prescriptive sense
this cleavage can be used to recover peptides that have undergone tryptophan crosslinking, although the yields drop
with increasing peptide length due to competitive cleavage of the amide bonds. The best conditions for cleavage involve
ꢀ
heating the dimeric peptides in dilute ethanolic HCl at 150 C in the presence of ten equivalents of ethanedithiol.
#
1998 Elsevier Science Ltd. All rights reserved.
Introduction
oxidized to a€ord ditryptophans, a new class of sym-
metrical homodimeric peptide crosslinks.11
Tryptophan is notorious among the amino acids for its
1
ability to cause problems during peptide synthesis. The
Steam distillation of skatole dimer from a solution of
dilute hydrochloric acid has been shown to produce
skatole, presumably by the reverse of the Mannich-type
dimerization shown in Scheme 1. We sought to develop
thermodynamic conditions that could facilitate trypto-
phan dimer cleavage (without removal of the product by
nucleophilicity of unprotected tryptophan leads to
alkylation by the cationic species which are abundant
under typical acidic peptide deprotection conditions. To
overcome problems of alkylation, special deprotection
cocktails have been formulated to include a variety of
nucleophiles that can scavenge benzylic and t-butyl
cations. These nucleophiles commonly include water,
dimethylsul®de, anisole, thiocresol, and ethanedithiol.1
Even in the absence of external electrophiles, trypto-
phan can undergo `self alkylation' by acid-promoted
Mannich dimerization.4±6 We have found that trypto-
phan dimers, once formed, can be cleaved under acidic
conditions at elevated temperatures. Amide bond
hydrolysis competes with tryptophan dimer cleavage, so
dilute acid gives the best yields.
9
distillation). Additionally we hoped to minimize amide
bond hydrolysis and thus provide a means of salvaging
intact peptides from product mixtures derived from
uncontrolled crosslinking of tryptophan sidechains.
±3
Results and Discussion
Preparation of substrates
Substrates were prepared by stirring tryptophan-con-
taining peptides in neat tri¯uoroacetic acid at room
temperature. This reaction leads to the formation of
diastereomeric tryptophan dimers, which are typically
isolable in about 25±75% yield after silica gel chroma-
tography or reverse-phase HPLC. For example, stirring
N-acetyltryptophan ethyl ester in anhydrous TFA under
nitrogen for 12 h leads to formation of dimers 2a and 2b
in 33% and 41% isolated yields, respectively [eq. (1)].
These dimers have the same relative indoline stereo-
chemistry, and this stereochemistry is trans,12 but we
have not yet determined which structure corresponds to
The potential for crosslinking of tryptophan sidechains
under acidic conditions was ®rst proposed by Elliott in
7
1
dimers was known.
952 ±long before the true structure of 3-alkylindole
±10
8
Tryptophan dimerization was
demonstrated over 20 years later by Shimonishi and co-
workers using Ac-Trp-OMe, Ac-Gly-Trp-OMe, and Ac-
6
Trp-Trp-OMe. While this reaction produces diastereo-
meric tryptophan dimers, they may be convergently
*
Corresponding author.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00071-6

9
76
B. Biggs et al./Bioorg. Med. Chem. 6 (1998) 975±981
tri¯uoroacetic acid was found to be reversible leading to
a 1.0:4.2:1.7 ratio of 1, 2a, and 2b, respectively. Trypto-
phan dimers are unstable to typical conditions13,14 used
ꢀ
for hydrolysis of proteins: 6 N aq HCl, 110 C, degas-
sed.15 Under these conditions, tryptophan dimer 2a is
eciently cleaved and hydrolyzed within 17 h to a€ord
monomeric tryptophan in over 90% yield. Even at
room temperature, about one third of the dimer is
cleaved by 6 N aq HCl within 10 h. The important
implication of these results is that tryptophan dimers
can not be detected by acidic hydrolysis of proteins;
their rapid cleavage makes them indistinguishable from
unmodi®ed tryptophan.
Scheme 1.
the low R isomer 2a and which structure corresponds to
f
the high R
f
isomer 2b (ethyl acetate/SiO
2
). The low R
f
ꢀ2†
isomer 2a was used to optimize conditions for the retro-
Mannich cleavage of tryptophan dimers without con-
comitant amide bond hydrolysis. Tryptophan dimers
are not symmetrical so intramolecular crosslinking (as
with Ac-Trp-Trp-OEt) is expected to lead to regio-
isomers as well as diastereomers. TFA cyclization of Ac-
Trp-Trp-OMe and tosylation of the indoline nitrogen
allowed the major regioisomer to be assigned to that which
6
results from attack of Trp H+ on Trp . For all of the
1
2
tryptophan dimers in this work, convergent oxidation to
the symmetrical ditryptophans using DDQ provided
con®rmation of the tryptophan dimer structure.11
Harnessing the retro-Mannich cleavage of tryptophan
dimers required that we develop conditions that would
retain the integrity of the amide bonds. Ethanol was
chosen as solvent for optimization of tryptophan dimer
cleavage to facilitate solubility and ease of substrate
handling [eq. (2), Table 1]. Also, on the assumption that
monomer and dimer might be in equilibrium, reactions
were run at low concentration (0.02 M) to favor mono-
mer over dimer. Tryptophan dimers are stable in anhy-
drous 1 M ethanolic HCl at room temperature (entry 1),
ꢀ
but at 78 C they undergo retro-Mannich cleavage
ꢀ
1†
(entry 2). Unfortunately, under these strongly acidic
conditions the mass balance was poor and monomer 3
was formed as the major product due to ethanolysis of
the amide bond. Since acidic ethanolysis of amide bonds
is ®rst order in [EtOH
2
+], we hoped to reduce this side
reaction by reducing the concentration of acid.16 The
combined yield of monomers 1 and 3 was highest with
0
HCl (entries 3 and 4). There was no reaction in dilute
.6 M HCl, but the mass balance was highest at 0.2 M
ꢀ
acid at 78 C (entry 5). Since thiols are known to
increase recovery of tryptophan during protein hydro-
lysis17 we explored their e€ect on tryptophan dimer
cleavage. It was found that addition of 1,2-ethanedithiol
(EDT) led to enhanced mass balance (entries 6±8).
Optimization of retro-Mannich cleavage conditions
In contrast to the initial report of Shimonishi, the
dimerization of tryptophan derivative 1 in anhydrous

B. Biggs et al./Bioorg. Med. Chem. 6 (1998) 975±981
977
Table 1. Tryptophan dimer cleavage versus amide hydrolysis in ethanol (eq. (2))
ꢀ
EDTa
Entry
Time (h)
T ( C)
[HCl] (M)
Recovered SM (%)
3 (%)
1 (%)
1
2
3
4
5
6
7
8
9
24
5
rt
78
1.00
1.00
0.60
0.20
0.01
0.20
0.20
0.20
0.20
0.20
0.20c
(±)
(±)
100
0
±
±
28
41
27
±
4
5
78
78
(±)
(±)
0
4
5
29
100
37
35
±
7
40
5
78
78
(±)
±
10 equiv.
100 equiv.
10 equiv.
(±)
10
9
13
14
5
78
78
16
1.5
1.5
1.5
10
17
±
(71b)
150
150
150
±
44
88 (99b)
64
1
1
0
1
10 equiv.
(±)
±
±
16
aEDT=ethanedithiol.
bHPLC yield (internal standard: p-nitrophenol).
cDegassed.
ꢀ
Table 2. Cleavage of tryptophan dimers in 0.2 M HCl/EtOH
ꢀ
When the temperature was increased to 150 C and 10
equiv. ethanedithiol was present, the retro-Mannich
cleavage was complete in less than 2 h, and monomer 1
could be isolated before ethanolysis of the amide bond
could occur (entries 9 and 10). Thiols are purported to
improve yields of tryptophan recovery through an anti-
oxidant e€ect.17±20 Partial con®rmation of this e€ect
came through repeating the cleavage in a sealed tube
with freeze-pump-thaw degassed solvent (entries 9 and
at 150 C
Substrate
Cleavage yield
1
1). This led to an improved yield of 1, but still did not
match the e€ect of added thiol. In general, to cleave
tryptophan dimers while minimizing amide bond hydro-
lysis one should heat at high temperatures for short
times in dilute acid (ca. 0.2 M) with EDT as an additive.
Variation of peptide
We next sought to investigate the generality of the opti-
mized conditions from Table 1, entry 10: 0.02 M sub-
strate, 0.2 M 1,2-ethanedithiol, 0.2 M ethanolic HCl,
ꢀ
sealed tube, 150 C, 1.5 h. The results are summarized in
Table 2. Importantly, both diastereomers 2a and 2b are
cleaved with equal ecacy using these conditions
Table 1, entry 10 and Table 2). Substrates with more
(
amide bonds gave lower isolated yields, presumably
due to competitive ethanolysis of the amide bonds.
Interestingly, the cyclic tryptophan dimer 4, which has
two amide bonds, gave comparable yields to tryptophan
dimers 5 and 6, which have twice as many amide bonds.
Yields are expected to decrease with increasing peptide
length, but the peptide sequence is also likely to in¯u-
ence the cleavage yield just as it does in the formation of
ditryptophans.11 Since there were no other major
products in these reaction mixtures, it is probable that
non-selective ethanolysis of the amide bonds occurs
both in the dimeric substrate and in the monomeric
product.

9
78
B. Biggs et al./Bioorg. Med. Chem. 6 (1998) 975±981
Reductive cleavage of tryptophan dimers
cleaved under typical conditions for protein hydrolysis
ꢀ
(
6 N HCl, 110 C), and are not revealed by genetic
The forcing conditions required for the cleavage of
tryptophan dimers led us to explore milder methods
based on kinetic trapping of monomeric tryptophan.
Since triethylsilane e€ectively reduces tryptophan in
TFA we investigated the reductive removal of trypto-
phan from the monomer/dimer equilibrium.2 Heating
sequence analysis, it is possible that tryptophan dimers
play some role in the post-translational modi®cation of
proteins but have simply gone undetected.
Experimental
ꢀ
tryptophan dimer 2b at 50 C in TFA with 10 equiv. of
triethylsilane established the monomer/dimer equili-
brium, but reduction was sluggish at this temperature.
Peptide substrates were synthesized using published
solution-phase BOC chemistry and DCC/HOBt cou-
plings; BOC deprotections were carried out with etha-
nolic HCl.
ꢀ
By raising the temperature to 75 C monomeric trypto-
phan was reduced slowly and selectively to dihydro-
tryptophan 8 over 12 h; no reduction of the tryptophan
dimer was observed. The crude product was oxidized
with DDQ in anhydrous dioxane to a€ord tryptophan
monomer 1 in a 74% overall yield from dimer 2b. While
the two step reductive cleavage/oxidation sequence
seems to work well, DDQ is unlikely to be compatible
with peptides that contain oxidatively sensitive amino
acids such as cysteine and methionine.
General procedure I for the Nꢁ-acetylation of peptides
To a solution of the corresponding hydrochloride salt in
pyridine (0.30 M) was added acetic anhydride (1.30
equiv.). The reaction was stirred at room temperature
for 12 h and then concentrated in vacuo. The residue
was dissolved in ethyl acetate and washed with 1 N HCl.
4
The organic layer was dried over MgSO and con-
centrated in vacuo. The crude product was puri®ed by
chromatography on silica gel.
N-Acetyl glycyltryptophan ethyl ester, Ac-Gly-Trp-OEt.
Glycyltryptophan ethyl ester, hydrochloride (5.88 g,
18.1 mmol) was acetylated according to general proce-
dure I to a€ord Ac-Gly-Trp-OEt (5.84 g, 98%) as a
ꢀ
white solid. Mp 148±150 C (CHCl ); R =0.47 (10%
MeOH/CHCl ); IR (KBr) 3599, 3425, 3259, 3062, 2927,
3
3
f
ꢀ
3†
745, 1647 cm 1; 1H NMR (500 MHz, DMSO-d
�
1
1
1
7
6
) d
0.88 (s, 1H), 8.24 (d, J=7.0 Hz, 1H), 8.05 (t, 5.3 Hz,
H), 7.48 (d, J=8.0 Hz, 1H), 7.33 (d, J=8.0 Hz, 1H),
.14 (d, J=2.0 Hz, 1H), 7.06 (t, J=7.3 Hz, 1H), 6.98 (t,
J=7.0 Hz, 1H), 4.49 (q, J=7.3 Hz, 1H), 3.99 (q,
J=7.0 Hz, 2H), 3.72 (dd, J=16.8, 5.8 Hz, 1H), 3.67 (dd,
J=16.8, 5.8 Hz, 1H), 3.13 (dd, J=14.0, 7.0 Hz, 1H),
3
.05 (dd, J=14.5, 8.0 Hz, 1H), 1.82 (s, 3H), 1.07 (t,
J=7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d
) d
71.8, 169.5, 169.0, 136.1, 127.1, 123.8, 121.0, 118.4,
17.9, 111.4, 109.2, 60.5, 53.1, 41.7, 27.1, 22.4, 13.9;
6
1
1
+
+
LRMS, CI , m/e (%) 331 (MH , 125), 215 (52), 159
+
Conclusion
(
15), 143 (20), 130 (100); HRMS (CI ) calcd for
In conclusion, we have shown that the Mannich cross-
linking of tryptophan sidechains, a side reaction in pep-
tide synthesis, may be reversed by heating a dilute
C
C
17
17
H
H
21
21
N
N
3
O
O
4
4
, 331.1532; found 331.1533. Anal. calcd for
: C, 61.60; H, 6.39; N, 12.69. Found: C,
3
61.65; H, 6.31; N, 12.65.
solution of peptide dimer in 0.2 M ethanolic HCl with
ꢀ
1
0 equiv. 1,2-ethanedithiol at 150 C in a sealed tube.
N-Acetyl tryptophanylglycine ethyl ester, Ac-Trp-Gly-
OEt. Tryptophanylglycine ethyl ester hydrochloride
(4.99 g, 16.0 mmol) was acetylated according to general
Ethanolysis also occurs under these conditions leading
to a reduction in the isolated yield of monomeric pep-
tide. Cleavage is also possible under reductive condi-
tions leading to partial kinetic trapping of the
tryptophan monomer. There have been no reports of
tryptophan dimers in naturally occurring proteins or
peptides. However, since tryptophan dimers are readily
procedure I to a€ord Ac-Trp-Gly-OEt (4.96 g, 96%) as
ꢀ
a white solid. mp 158±160 C (CHCl
MeOH/CHCl ); IR (KBr) 3388, 3292, 3070, 2927, 2850,
3
); R
f
=0.54 (10%
3
1730 cm 1; 1H NMR (500 MHz, DMSO-d
�
) d 10.80 (s,
1H), 8.46 (t, J=5.8 Hz, 1H), 8.09 (d, J=8.5 Hz, 1H),
6

B. Biggs et al./Bioorg. Med. Chem. 6 (1998) 975±981
979
7
.60 (d, J=8.0 Hz, 1H), 7.32 (d, J=8.5 Hz, 1H), 7.14 (d,
the remaining mixture was separated by silica gel chro-
matography (33% acetone/chloroform) to a€ord dimer
2a (3.25 g, 33%) and dimer 2b (4.05 g, 41%). 2a: mp
J=1.5 Hz, 1H), 7.06 (t, J=7.5 Hz, 1H), 6.98 (t,
J=7.0 Hz, 1H), 4.57 (m, 1H), 4.09 (q, J=7.0 Hz, 2H),
ꢀ
3.81 (m, 2H), 3.17 (dd, J=14.0, 4.3 Hz, 1H), 2.89 (dd,
J=14.5, 9.5 Hz, 1H), 1.77 (s, 3H), 1.19 (t, J=6.8 Hz,
216±218 C (EtOAc); R
f
=0.35 (10% acetone/chloro-
form); IR (KBr) 3296, 3077, 2992, 1743, 1618,
H); 13C NMR (125 MHz, DMSO-d
70.7, 137.6, 129.0, 124.7, 123.7, 121.2, 120.2, 112.7,
1556 cm ; H NMR (500 MHz, DMSO-d ) d 10.91 (s,
� 1
1
3
1
1
6
) d 172.9, 171.6,
6
1H), 8.32 (d, J=7.0 Hz, 1H), 8.18 (d, J=7.5 Hz, 1H),
7.45 (d, J=8.0 Hz, 1H), 7.25 (d, J=8.0 Hz, 1H), 7.15 (d,
J=7.5 Hz, 1H), 7.02 (ddd J=16.5, 7.1, 1.5 Hz, 2H), 6.94
(t, J=7.3 Hz, 1H), 6.67 (t, J=7.5 Hz, 1H), 6.61 (d,
J=8.0 Hz, 1H), 6.00 (d, J=3.0 Hz, 1H), 5.97 (dd,
J=9.3, 3.3 Hz, 1H), 4.53 (q, J=6.5 Hz, 1H), 4.26 (q,
J=6.5 Hz, 1H), 3.96 (m, 4H), 3.46 (q, J=6.5 Hz, 1H),
+
12.0, 62.9, 55.2, 42.8, 29.6, 24.7, 15.5; LRMS, FAB ,
+
m/e (%) 332 (MH , 100), 273 (78), 230 (38), 202 (32),
1
3
29 (62); HRMS (FAB+) calcd for
31.1532; found 331.1533. Anal. calcd for C H N O :
C
H
21
17
3
N O
4
1
7
21
3
4
C, 61.60; H, 6.39; N, 12.69. Found C, 61.61; H, 6.45; N,
2.57.
1
3
7.5 Hz, 1H), 2.09 (m, 1H), 1.98 (m, 1H), 1.80 (s, 3H),
.17 (dd, J=14.25, 6.25 Hz, 1H), 3.08 (dd, J=14.5,
N-Acetyl tryptophyltryptophan ethyl ester, Ac-Trp-Trp-
OEt. Tryptophanyltryptophan ethyl ester hydro-
chloride (7.72 g, 17.8 mmol) was acetylated according to
1.60 (s, 3H), 1.05 (t, J=3.75 Hz, 3H), 1.03 (t, J=3.5 Hz,
3H); 13C NMR (125 MHz, CDCl
3
) d 172.1, 171.9, 169.2,
general procedure I to a€ord Ac-Trp-Trp-OEt (7.66 g,
ꢀ
169.2, 150.6, 136.9, 135.7, 130.5, 127.9, 127.7, 123.5,
120.9, 118.2, 118.0, 117.6, 111.1, 108.6, 107.1, 61.4, 60.5,
60.4, 53.5, 50.6, 45.5, 35.1, 26.3, 22.3, 21.9, 13.8, 13.8;
9
8%) as a white solid. mp 98±150 C (CHCl
3
); R
f
=0.48
(
1
3
10% MeOH/CHCl ); IR (KBr) 3403, 3055, 2927, 1734,
�
1
1
+
+
654 cm ; H NMR (500 MHz, DMSO-d ) d 10.90 (s,
LRMS, FAB , m/e (%) 549 (MH , 100), 520 (45), 257
+
6
1
H), 10.80 (s, 1H), 8.43 (d, J=7.0 Hz, 1H), 8.02 (d,
(70), 169 (66), 130 (70); HRMS (FAB ) calcd for
J=8.5 Hz, 1H), 7.61 (d, J=8.0 Hz, 1H), 7.49 (d,
J=7.5 Hz, 1H), 7.33 (t, J=8.5 Hz, 2H), 7.16 (d,
J=2.0 Hz, 1H), 7.11 (d, J=2.0 Hz, 1H), 7.06 (q,
J=7.5 Hz, 2H), 6.98 (q, J=7.2 Hz, 2H), 4.61 (m, 1H),
C
C
30
30
H
H
36
36
N
N
4
O
O
6
6
, 548.2635; found 548.2652. Anal. calcd for
: C, 65.66; H, 6.62; N, 10.22. Found: C,
4
65.40; H, 6.57; N, 10.16.
ꢀ
4
(
.52 (q, J=7.0 Hz, 1H), 4.00 (q, J=7.3 Hz, 2H), 3.13
m, 3H), 2.87 (m, 1H), 1.75 (s, 3H), 1.07 (t, J=7.5 Hz,
2b. Mp 103±105 C (CHCl
chloroform); IR (KBr) 3349, 3048, 2971, 1723, 1631,
1529 cm ; 1H NMR (500 MHz, DMSO-d
3
); R
f
=0.44 (33% acetone/
H); 13C NMR (125 MHz, DMSO-d
69.1, 136.1, 136.0, 127.3, 127.1, 123.7, 123.5, 121.0,
) d 172.0, 171.7,
� 1
) d 10.77 (s,
3
1
1
1
6
6
1H), 8.29 (m, 2H), 7.47 (d, J=8.0 Hz, 1H), 7.28 (d,
J=8.0 Hz, 1H), 7.07 (d, J=7.0 Hz, 1H), 7.02 (m, 2H),
6.95 (t, J=7.3 Hz, 1H), 6.63 (t, J=6.5 Hz, 1H), 5.89 (s,
1H), 4.83 (d, J=8.5 Hz, 1H), 4.52 (m, 2H), 4.02 (m, 2H),
3.97 (m, 2H), 3.40 (q, J=6.5 Hz, 1H), 3.19 (dd, J=14.1,
4.8 Hz, 1H), 3.06 (dd, J=14.1, 9.2 Hz, 1H), 2.14 (m,
1H), 1.95 (m, 1H), 1.79 (d, J=1.5 Hz, 3H), 1.75 (d,
J=2.0 Hz, 3H), 1.08 (dt, J=14.0, 7.0 Hz, 3H), 1.04 (dt,
20.8, 118.5, 118.4, 118.2, 118.0, 111.4, 111.3, 110.2,
09.3, 60.4, 53.2, 53.0, 27.7, 26.9, 22.5, 13.9; LRMS, CI,
+
m/e (%) 460 (MH , 100), 229 (32), 215 (70), 186 (12),
+
155 (13), 145 (18), 130 (100); HRMS (CI ) calcd for
C H N O , 460.2110; found 460.2111. Anal. calcd for
26
28
4
4
C
6
26
H
28
N
4
O
7.48; H, 6.06; N, 11.98.
4
: C, 67.79; H, 6.13; N, 12.17. Found C,
J=14.5, 7.5 Hz, 3H); 13C NMR (125 MHz, CDCl ) d
3
General procedure II for the formation of tryptophan di-
mers. The corresponding tryptophan peptides were dis-
solved in tri¯uoroacetic acid (0.30 M) and stirred at
room temperature for 12 h. The reaction mixture was
concentrated in vacuo, taken up in ethyl acetate and
washed with sat. aq. sodium bicarbonate. The aqueous
phase was extracted with ethyl acetate and the combined
172.3, 172.2, 169.6, 169.5, 151.0, 137.5, 135.8, 129.7,
127.8, 127.7, 124.1, 120.9, 118.3, 117.9, 117.4, 111.3,
108.8, 107.3, 60.7, 60.5, 59.8, 53.6, 49.8, 45.3, 35.7,
+
26.3, 22.4, 22.2, 13.9, 13.9; LRMS, FAB , m/e (%)
+
549 (MH , 100), 404 (43), 259 (26), 154 (37), 130
+
(31); HRMS (FAB ) calcd for C H N O , 548.2635;
6
30
36
4
found 548.2627. Anal. calcd for C30
35 4 6
H N O : C,
organic layers were dried over MgSO
4
. Solvent was
65.66; H, 6.62; N, 10.22. Found: C, 65.43; H, 6.58; N,
10.17.
removed in vacuo and the diastereomeric tryptophan
dimers were separated from starting material by chro-
matography on silica gel or reverse phase HPLC (20±
Formation of cyclic tryptophan dimer (4). Ac-Trp-Trp-
OEt (0.20 g, 0.43 mmol) was cyclized with tri¯uoroacetic
acid according to general procedure II to a€ord trypto-
phan dimer 4 as a mixture of diastereomers (0.060 g,
1
2
00% MeCN/H O over 30 min).
Formation of tryptophan dimers 2a and 2b. N-Acetyl
tryptophan ethyl ester 1 (9.83 g, 35.8 mmol) was dimer-
ized in tri¯uoroacetic acid according to general proce-
dure II. The bulk of dimer 2a was partially crystallized
from the mixture of diastereomers (ethyl acetate) and
f
31%). R =0.45 (10% methanol/chloroform); LRMS,
+
+
FAB , m/e (%) 461 (MH , 100), 442 (27), 271 (33), 259
+
(79), 245 (76), 130 (100); HRMS (FAB ) calcd for
C H N O
26 28 4 4
, 460.2110; found 461.2196 (MH+).

9
80
B. Biggs et al./Bioorg. Med. Chem. 6 (1998) 975±981
ꢀ
Formation of tryptophan dimer [Ac-Gly-Trp-OEt]
2
(5).
at 150 C for 1.5 h the mixture was allowed to cool to
room temperature and concentrated in vacuo. The
crude product was puri®ed by chromatography on silica
gel.
Ac-Gly-Trp-OEt (0.50 g, 1.50 mmol) was dimerized
with tri¯uoroacetic acid according to general procedure
II to a€ord tryptophan dimer 5 as a mixture of diaster-
eomers (white solid: 0.36 g, 72%). R =0.10 (5% metha-
f
+
+
nol/chloroform); LRMS, FAB , m/e (%) 663 (MH ,
00), 596 (11), 549 (13), 523 (9), 509 (5); HRMS
FAB+) calcd for C H N O , 662.3063; found
Cleavage of 2a. Tryptophan dimer 2a (0.10 g,
0.18 mmol) was cleaved according to general procedure
III to a€ord 1 (0.088 g, 88%) as a white solid.
1
(
3
4
42
6
8
6
63.3142 (MH+).
Cleavage of 2b. Tryptophan dimer 2b (0.10 g,
0.18 mmol) was cleaved according to general procedure
III to a€ord 1 (0.089 g, 89%) as a white solid.
Formation of tryptophan dimer [Ac-Trp-Gly-OEt]2 (6).
Ac-Trp-Gly-OEt (0.20 g, 0.60 mmol) was dimerized
with tri¯uoroacetic acid according to general procedure
II to a€ord tryptophan dimer 6 as a mixture of diaster-
2
Cleavage of [Ac-Gly-Trp-OEt] (5). Tryptophan dimer 5
eomers (white solid: 0.07 g, 33%). R =0.11, 0.14 (5%
f
(0.10 g, 0.15 mmol) was cleaved according to general
procedure III to a€ord Ac-Gly-Trp-OEt (0.04 g, 40%).
+
methanol/chloroform); LRMS, FAB , m/e (%) 663
N
(
6
MH+, 100); HRMS (FAB+) calcd for C34
62.3063; found 663.3150 (MH+).
H
42
6
O
8
,
Cleavage of [Ac-Trp-Gly-OEt] (6). Tryptophan dimer 6
2
(0.10 g, 0.15 mmol) was cleaved according to general
procedure III to a€ord Ac-Trp-Gly-OEt (0.054 g, 54%).
Formation of tryptophan dimers [Ac-Gly-Trp-Gly-OEt]
2
(7). Ac-Gly-Trp-Gly-OEt (0.10 g, 0.26 mmol) was
dimerized with tri¯uoroacetic acid according to general
procedure II to a€ord tryptophan dimer 7 as a mixture
Cleavage of cyclic tryptophan dimer 4. Cyclic trypto-
phan dimer 4 (0.10 g, 0.22 mmol) in ethanol was cleaved
with concentrated HCl and ethanedithiol according to
general procedure III to a€ord Ac-Trp-Trp-OEt (0.05 g,
50%).
f
of diastereomers (white solid: 0.08 g, 77%). R =0.26,
+
0
.32 (10% methanol/chloroform); LRMS, FAB , m/e
+
(
(
%) 777 (MH , 100), 577 (12), 549 (19), 535 (14), 518
+
12), 509 (7); HRMS (FAB ) calcd for C38
76.3493; found 777.3566 (MH+).
48 8 10
H N O ,
7
Cleavage of [Ac-Gly-Trp-Gly-OEt]2 (7). Tryptophan
dimer 7 (0.08 g, 0.10 mmol) in ethanol was cleaved with
concentrated HCl and ethanedithiol to a€ord Ac-Gly-
Trp-Gly-OEt (0.025 g, 33%).
Reductive cleavage of tryptophan dimer 2b
To solution of tryptophan dimer 2b (0.50 g,
a
.91 mmol) in tri¯uoroacetic acid (45 mL) was added
0
triethylsilane (0.73 mL, 4.54 mmol) dropwise at 0 C.
Acknowledgements
ꢀ
ꢀ
ꢀ
The mixture was stirred at 0 C for 0.5 h then at 70 C
for 12 h followed by concentration in vacuo. The crude
product was oxidized without further puri®cation.
This work is supported by the the Petroleum Research
Fund, Camille and Henry Dreyfus Foundation, Glaxo-
Wellcome, NSF (CHE-9623903) and the NIH
(
GM54523).
The crude product mixture was dissolved in 1,4-dioxane
20 mL) and oxidized with DDQ (0.25 g, 1.10 mmol).
After 4 h of stirring at room temperature, ethyl acetate
was added (30 mL) and the mixture was washed with
sat. aq. sodium bicarbonate (10Â15 mL) until the
washes were no longer colored. The organic phase was
(
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1
1
2
. Pearson, D. A.; Blanchette, M.; Baker, M. L.; Guindon, C.
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4
and concentrated in vacuo. Chroma-
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7
1
4
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1
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