Problems associated with use of the N(in)-cyclohexyloxycarbonyl (Hoc) group for tryptophans

Article abstract of DOI:10.1016/S0040-4039(00)01156-4

During HF reaction in the presence of anisole or p-cresol, a side reaction was observed associated with use of the N(in)-cyclohexyloxycarbonyl (Hoc) group for tryptophan (Trp). The structure of the modified Trp and the HF conditions for suppressing this side reaction are described. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01156-4

Tetrahedron Letters 41 (2000) 6839±6842
Problems associated with use of the
Nin-cyclohexyloxycarbonyl (Hoc) group for tryptophans
Hideki Nishio, Yuji Nishiuchi,* Tatsuya Inui, Kumiko Yoshizawa-Kumagaye
and Terutoshi Kimura
Peptide Institute Inc., Protein Research Foundation, 4-1-2 Ina, Minoh-shi, Osaka 562-8686, Japan
Received 8 June 2000; accepted 7 July 2000
Abstract
During HF reaction in the presence of anisole or p-cresol, a side reaction was observed associated with
use of the Nin-cyclohexyloxycarbonyl (Hoc) group for tryptophan (Trp). The structure of the modi®ed Trp
and the HF conditions for suppressing this side reaction are described. # 2000 Elsevier Science Ltd. All
rights reserved.
Keywords: HF reaction; side reaction; Trp modi®cation.
In the synthesis of tryptophan (Trp)-containing peptides by Boc chemistry, the functional
group of the indole ring must be protected by appropriate protecting groups to avoid oxidation
and alkylation during repetitive acid treatment such as with TFA.1 For this purpose, the formyl
(For) group2 has been widely employed. However, the For group makes necessary the use of thiol
at the ®nal HF reaction to remove it3 and is also susceptible under conditions using basic
nucleophiles4 (e.g. morpholine or piperidine). To overcome these disadvantages with the For
group, we developed the cyclohexyloxycarbonyl (Hoc) group5 as a base-resistant Nin-protecting
group. The Hoc group is stable under general conditions during assembly of the protected
peptide and is readily cleaved by HF without the need to resort to the use of thiol. The utility of
the Hoc group has been demonstrated by synthesizing various biologically active Trp-containing
peptides6 and proteins.7 However, closer examination of products by HPLC and ESI MS
revealed that a signi®cant side reaction had occurred during HF cleavage of Trp(Hoc)-containing
peptides in the presence of p-cresol or anisole as a scavenger. Each side product arising from a
modi®cation of the Trp residue had a MW 108 higher than that of the desired peptide, although
the extent of this side reaction seemed to display sequence dependency. In the present communication,
we report the structure of the side products and the conditions which can suppress this side
reaction with the Trp residue.
* Corresponding author. Tel: +81-727-29-4121; fax: +81-727-29-4124; e-mail: yuji@prf.or.jp
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01156-4

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In model experiments using Ac-Trp(X)-NH2 [X=Hoc, benzyloxycarbonyl (Z), 2,4-dimethylpent-
3-yloxycarbonyl (Doc)8 or no protecting group], the same side products were obtained when
derivatives having carbamate-type Nin-protecting groups were treated with HF in the presence of
p-cresol. On the other hand, no product with this side reaction was observed in the case of
treatment of Ac-Trp-NH2 in the presence of p-cresol. The Nin-carboxylate intermediate generated
from a carbamate-type Nin-protecting group might participate in this side reaction. These results
showed that this side reaction is inherent to the carbamate-type Nin-protecting groups. Next, in
order to elucidate this side reaction with the Trp residue, the side products obtained after treating
Ac-Trp(Hoc)-NH2 1 with HF/p-cresol (v/v = 8/2, ^5ꢀC for 1 h) were isolated and characterized
by ESI MS (Fig. 1).
Figure 1. HPLC pro®le of products obtained after treatment of Ac-Trp(Hoc)-NH2 1 with HF/p-cresol
Products 4 and 6 corresponded to two steric isomers of the Trp-dimer as reported previously.9
The UV spectrum (in 1% AcOH) of products 2 and 5 gave identical pro®les (lmax 286, 268, 262
nm) indicating modi®cation of the indole ring in comparison with that of the desired product 3
[lmax 288, 280, 273(sh) nm]. The structures of 2 and 5, whose MW were 108 mass units higher
than that (246.1) of 3, were determined by NMR10 as shown in Scheme 1. This revealed that the
Trp residue was preferentially converted to two diastereoisomers of 2-acetylamino-3-[2-(2-
hydroxy-5-methylphenyl)-2,3-dihydro-1H-indol-3-yl]-propionamide, i.e., 2-[20-(p-cresol)]-2,3-
dihydrotryptophan, in which two centers of asymmetry were, in principle, generated at the 2- and
3-positions to produce four steric isomers. With regard to the orientation of p-cresol adducts 2
and 5, the coupling constant between C(2)-H and C(3)-H could not explain the distinction
between the trans- and the cis-conformation since these adjacent protons are located on a ®ve-
membered ring (JH2,H3 = 5.27 for 2, JH2,H3 = 5.26 for 5). Therefore, we measured NOE using
two-dimensional rotating frame experiments (ROESY) to obtain information about the distance
between C(2)-H and C(3)-H and that between C(2)-H and b-CH2. In both 2 and 5, the latter was
found to be apparently closer than the former.11 From these results, 2 and 5 were deduced to be
the trans-isomers. As for the treatment of the Trp(Hoc)-containing peptides with HF in the pre-
sence of anisole instead of p-cresol, the same side reaction with the Trp residue also occurred to
produce the trans-anisole adducts corresponding to 2 and 5. As anisole has two possible positions

6841
Scheme 1.
for substitution, i.e. the ortho- and para-positions, the anisole adducts were identi®ed as four
steric trans-isomers having a MW 108 higher than that of the desired product (data not shown).
Next, we investigated the HF conditions for Ac-Trp(Hoc)-NH2 1 to suppress the modi®cation
of the Trp residue. In the practical peptide synthesis, the use of thiol such as 1,4-butanedithiol
(BDT) during the HF reaction was found to signi®cantly reduce the extent of this side reaction
although the Hoc group could be removed by HF without any thiol (Table 1). However, the use
of thiol in the HF reaction often causes handling problems such as unpleasant odors. On the
other hand, when treating 1 with HF without any scavenger, the low yield of 3 was accompanied
by a mixture of Trp derivatives substituted by cyclohexyl groups at multiple positions. Therefore,
if an ecient scavenger to replace p-cresol or anisole is available, it would be one solution for this
problem associated with the use of the Hoc group for the Trp residue. As the Fmoc group is
known to be capable of capturing cations generated from the side-chain protecting groups with
Boc chemistry during the HF reaction,12 Fmoc-amino acid (Fmoc-Leu) was tested as a scavenger.
The desired product 3 was obtained in a quantitative yield without any adduct between Trp and
Fmoc-Leu.
Table 1
Products obtained after treatment of Ac-Trp(Hoc)-NH2 1 with HF
In summary, we have identi®ed a side reaction associated use of the Hoc group for the Trp
residue during HF cleavage of Trp(Hoc)-containing peptides in the presence of p-cresol or
anisole. This side reaction could be completely suppressed by performing the HF reaction in the
presence of Fmoc-Leu as a scavenger.

6842
Acknowledgements
We wish to thank Dr. Toshimasa Yamazaki of the National Institute of Agrobiological
Resources for the NMR measurements.
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1
10. Product 2: H NMR (in DMSO-d6 270 MHz) ꢀ 1.74 (s, 3H, COCH3), 2.01 (m, 2H, b-CH2), 2.13 (s, 3H, p-cresol
CH3), 3.57 (m, 1H, indoline H3), 4.23 (m, 1H, a-CH), 4.79 (d, J=5.27, 1H, indoline H2), 6.75 (d, J=7.92, 1H, p-
cresol H60), 6.94 (d, J=7.95, 1H, p-cresol H50), 6.96 (s, 1H, p-cresol H30), 6.97 (s, 1H, CONH2), 6.99 (d, J=7.26,
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(d, J=7.26, 1H, indoline H4), 7.99 (d, J=7.25, 1H, Ac-NH-). Product 5: 1H NMR (in DMSO-d6 270 MHz) ꢀ 1.81
(s, 3H, COCH3), 2.00 (m, 2H, b-CH2), 2.12 (s, 3H, p-cresol CH3), 3.22 (m, 1H, indoline H3), 4.47 (m, 1H, a-CH),
4.75 (d, J=5.26, 1H, indoline H2), 6.78 (d, J=8.57, 1H, p-cresol H60), 6.93 (d, J=7.26, 1H, indoline H7), 6.94 (d,
J=8.57, 1H, p-cresol H50), 6.94 (s, 1H, p-cresol H30), 6.94 (t, J=7.26, 1H, indoline H5), 7.03 (s, 1H, CONH2), 7.18
(t, J=7.26, 1H, indoline H6), 7.24 (s, 1H, CONH2), 7.26 (d, J=7.26, 1H, indoline H4), 8.09 (d, J=8.58, 1H, Ac-
NH-).
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geminal methylene protons, which is calculated to be 1.76 A, was used for rkl as a standard.
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