Cyclohexyl Chloroformate

[13248-54-9]  · C7H11ClO2  · (162.62)

(cyclohexyloxycarbonyl (Hoc); amine and indole protecting group for peptide synthesis)

Alternate Name: carbonochloridic acid cyclohexyl ester; cyclohexyloxycarbonyl chloride; chloroformic acid cyclohexyl ester.

Analysis of Reagent Purity: 1H NMR.

Form Supplied in: colorless liquid; not commercially available.

Solubility: soluble in most organic solvents.

Handling, Storage, and Precautions: decomposes to CO2 and cyclohexyl chloride on prolonged storage. Store in a refrigerator. Bottles may develop pressure and should be cooled before opening. Use in a fume hood. Irritating to skin, eyes, mucous membranes.

Physical Data: bp 38-44 °C 2 mmHg;1 n20D 1.4587.1

Preparative Methods: a 1 L round-bottomed flask equipped with a delivery tube extending to the bottom of the flask, a pressure-equalizing addition funnel and a cold-finger condenser cooled with dry ice-acetone were placed in an ice bath. The exit tube of the condenser was connected to a CaCl2 drying tube and suitable trap for phosgene and HCl. Phosgene was introduced into the flask until 109 g (1.1 mol) has been condensed. The reaction flask is stirred while 100 g (1 mol) of cyclohexyl alcohol was added through the dropping funnel at a rate to keep the mixture in gentle reflux for 1 h. The flask was allowed to stand at room temperature with stirring for 2 h. The solution was then concentrated under reduced pressure at room temperature to remove HCl and the excess phosgene. The residue may be used without further purification.

Note: Phosgene is a highly toxic gas; use in a fume hood.

Introduction

The cyclohexyloxycarbonyl (Hoc) group was originally developed by McKay and Albertson as acid labile Na-amino protection used for peptide synthesis.2 The reagent used for the introduction of the Hoc group, cyclohexyl chlorofomate, is easily prepared from inexpensive starting materials, cyclohexyl alcohol, and phosgene (eq 1).

In elongating peptide chains properly, there must be a highly compatibility between the protecting groups for Na-amino function and amino acid side-chain functional groups. The Na-Hoc group is readily cleaved by strong acids such as hydrogen bromide (HBr) in acetic acid2 or liquid hydrogen fluoride (HF),3 although most side-chain protecting groups are also removed under these conditions. When compared with other Na-amino protecting groups commonly used for t-butyloxycarbonyl or benzyloxycarbonyl (Z)-mediated peptide synthesis, however, the Boc and the Z groups can be selectively removed by much milder conditions such that the side-chain protecting groups remain intact. Therefore, the Hoc group has not gained the practical application for Na-amino protection since it offers no advantage over the Boc and Z groups. However, recently the use of Hoc group has been used as the Nin-protecting group for the synthesis of tryptophan (Trp)-containing peptides4 and also as another orthogonal amino-protecting group in Boc chemistry.5

Hoc Group as a Base-Resistant Nin-Protecting Group for Trp

The Hoc group, which is compatible with Boc-mediated peptide synthesis, was introduced to the indole moiety of Trp to avoid indole oxidation and alkylation during repetitive acid treatment such as with trifluoroacetic acid (TFA).4,6 The Hoc group was introduced by acylation of Boc-Trp-OPac (Pac = phenacyl) or Boc-Trp-OBzl using cyclohexyl chloroformate in dichloromethane in the presence of pulverized NaOH and a catalytic amount of tetra-n-butyl ammonium hydrogensulfate (TBAHS) at room temperature for 1 h. Removal of the Pac or Bzl ester by using zinc dust in acetic acid or catalytic hydrogenation gave the Trp derivative (eq 2).

In contrast with the widely employed formyl protecting group for Trp,7 the Hoc group is readily cleaved by HF without resorting to the use of thiols and is also far more stable under conditions using basic nucleophiles (e.g. morpholine and piperidine)4 (Table 1).

When synthesizing Trp-containing peptides by the Boc-mediated peptide synthesis, therefore, the Nin-Hoc group is applicable not only to the standard solid-phase peptide synthesis but also to the preparation of protected peptide segments on base labile linkers such as an N-[9-(hydroxymethyl)-2-fluorenyl]succinamic acid (HMFS) linker.8 The HMFS linker is designed to release fully protected segments with a free a-carboxyl group by treatment with 20% morpholine in DMF. The Hoc group on Trp is completely stable under basic conditions for detachment of the segments from the HMFS resin. The protected segments thus obtained are used for the subsequent segment condensation reaction performed using either solution or solid phase method. Employing this procedure based on performing segment assembly on the solid support and segment condensation in solution, the synthesis of muscarinic toxin 1 (a 66-residue peptide)9 and green fluorescent protein (a 238-residue protein)10 was reported. With accumulation of experiments employing the Nin-Hoc group, however, it became apparent that modification of Trp associated with use of this group produces two trans-isomers of 2-[2-(p-cresol)]-2,3-dihydrotryptophan (1) during the HF reaction in the presence of p-cresol as a scavenger.11

This side reaction for Trp proved to be inherent to the carbamate-type Nin-protecting groups (e.g. Hoc, Z and 2,4-dimethylpent-3-yloxycarbonyl12). The use of thiols such as butane-1,4-dithiol during the HF reaction was found to almost completely avoid this side reaction, although the Hoc group could be removed by HF without any thiol.

Hoc Group as Another Orthogonal Amino-Protecting Group in Boc Chemistry

In Boc-mediated peptide synthesis, the Boc group is used as Na protection in combination with benzyl (Bzl)/cyclohexyl (Hex)-based side-chain protecting groups. Thus, the orthogonality of protecting scheme consists in the difference in reaction rate when acidolysis is used to remove different classes of protecting groups: generally TFA and HF are employed for removal of the Boc and the side-chain protecting groups, respectively. Regarding removal of the side-chain protecting groups, Bzl-based groups can be cleaved by 1M trimethylsilyl trifluoromethanesulphonate (TMSOTf)-thioanisole/TFA mixture13 to which Hex-based groups are stable. Therefore, free amino and/or carboxyl groups protected by Bzl-based groups are available prior to cleaving Hex-based protecting groups. By taking advantage of the difference in their removability under acidic conditions, a possibility for the assembly of cyclic lactam peptides was demonstrated.5

Related Reagents.

2-(t-Butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON), t-butylchloroformate (Boc-Cl), benzyl chloroformate.


1. Fourneau, M. E.; Montaigne, M.; Puyal, J., J. Anales Soc. Espanol Fis. Quim. 1920, 73, 323.
2. McKay, F. C.; Albertson, N. F., J. Am. Chem. Soc. 1957, 79, 4686.
3. Sakakibara, S.; Shimonishi, Y.; Kishida, Y.; Okada, M.; Sugihara, H., Bull. Chem. Soc. Jpn 1967, 40, 2164.
4. Nishiuchi, Y.; Nishio, H.; Inui, T.; Kimura, T.; Sakakibara, S., Tetrahedron Lett. 1996, 37, 7529.
5. Mezõ, G.; Mihala, N.; Kóczán, G.; Hudecz, F., Tetrahedron 1998, 54, 6757.
6. Jaeger, E.; Thamm, P.; Knof, S.; Wünsch, E.; Löw, M.; Kisfaludy, L., Hoppe-Seyler's Z. Physiol. Chem. 1978, 359, 1617.
7. Tam, J. P.; Heath, W. F.; Merrifield, R. B., J. Am. Chem. Soc. 1983, 105, 6442.
8. Rabanal, F.; Giralt, E.; Albericio, F. Tetrahedron 1995, 51, 1449.
9. Nishiuchi, Y.; Nishio, H.; Inui, T.; Bódi, J.; Kimura, T., J. Peptide Sci. 2000, 6, 84.
10. Nishiuchi, Y.; Inui, T.; Nishio, H.; Bódi, J.; Kimura, T.; Tsuji, F.I.; Sakakibara, S., Proc. Natl. Acad. Sci. USA 1998, 95, 13549.
11. Nishio, H.; Nishiuchi, Y.; Inui, T.; Yoshizawa-Kumagaye, K.; Kimura, T., Tetrahedron Lett. 2000, 41, 6839.
12. Karlström, A.; Undèn, A., Chem. Commun. 1996, 1471.
13. Fujii, N.; Otaka, A.; Ikemura, O.; Akaji, K.; Funakoshi, S.; Hayashi, Y.; Kuroda, Y.; Yajima, H. J., Chem. Soc., Chem. Commun. 1987, 274.

Yuji Nishiuchi

Peptide Institute Inc., Osaka, Japan



Copyright 1995-2000 by John Wiley & Sons, Ltd. All rights reserved.