4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) Linker

[136849-75-7]  · C12H16O5  · (MW 240.25)

(reagent used as a hyperacid sensitive linker in solid-phase synthesis, compatible with Fmoc/tBu solid-phase peptide synthesis)

Physical Data: mp 83-86°C.

Solubility: soluble in N-methyl pyrrolidone, DMF, other organic solvents.

Form Supplied in: commercially available as 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (1) or as prefunctionalized resins, in the form of 4-[4-(hydroxymethyl)-3-methoxyphenoxy] butanoic acid benzhydrylamine resin (HMPB-BHA) (2) and 4-[4-(hydroxymethyl)-3-methoxyphenoxy]butanoic acid 4-methyl benzhydrylamine resin (HMPB-MBHA) (3).

Handling, Storage, and Precautions: no special precautions. Keep cool and dry. Store at 8 °C.

Preparation of 1 and Attachment to a Solid Support

Linker 1 is conveniently prepared from 4-hydroxy-2-methoxybenzaldehyde in three steps.1 The linker may be coupled to any aminomethylated resin using standard coupling methods.2,3 The linker has also been coupled to dendrimer3 and TentaGel4 resins. Fmoc amino acids can be esterified onto the resulting HMPB functionalized resin using 2,6-dichlorobenzoyl chloride5 or standard DIC/DMAP conditions.3 Alcohols can be immobilized onto the HMPB functionalized resin under Mitsunobu conditions.6

Cleavage of Peptides from the HMPB Linker

The inclusion of the additional two methylene units in the carboxylic acid chain renders the HMPB linker 1 to be approximately 30 times more acid labile than the analogous 4-hydroxymethyl-3-methoxyphenoxy acetic acid linker introduced by Sheppard and Williams.7,8 This offers the potential that peptides attached to the linker can be easily cleaved by treatment with mildly acidic solutions such that common, acid susceptible amino acid side-chain protecting groups are retained on the liberated peptide fragments,9 the only exception being His(Trt) that in practice often loses small amounts of the trityl protecting group (although reports of this vary). It has been reported that the trityl protecting group may be selectively cleaved by mild acid treatment in the presence of other acid-sensitive functionalities.10 Conditions (0.1 M HOAt, 0.12 M Me3SiCl in 2,2,2-trifluoroethanol) suitable for the quantitative removal of a Na trityl unit of a resin-bound peptide using the 3-(4-hydroxymethylphenoxy)propionic acid linker were found to be too harsh for the HMPB handle as evidenced by the partial release of the peptide from the solid support.10 A more general approach for alkoxybenzyl ethers was found to be 0.2% TFA, 1% H2O in CH2Cl2, resulting in the desired cleavage without premature release of the peptide. The HMPB linker 1 has been successfully exploited in the preparation of peptides using a fragment-coupling approach, which conveniently combines the solid-phase synthesis of fully protected peptide units with the solution-phase union of these fragments.9 This fragment condensation methodology has been fruitfully applied to the synthesis of human calcitonin-(1-33), human neuropeptide Y (NPY), and the 230-249 sequence of mitogen-activated 70K S6 kinase.9 This method also has been used in the preparation of repetitive protein domains, protected peptide fragments being synthesized on solid-phase using Fmoc peptide synthesis.11 Interestingly the acid sensitive trityl group was successfully used to protect the side chain of histidine, which survived the cleavage protocol (1% TFA in CH2Cl2) to release the protected peptide from HMPB-MBHA (3) resin. Peptide couplings were performed using HATU/HOAt/i-Pr2NEt with isolated overall yields in excess of 80%.

Synthesis of Cyclic Peptides

Head to tail cyclization of tyrosine-containing peptides on the solid-phase using Fmoc chemistry has been used for the preparation of cyclic peptides.6 The phenolic group of the tyrosine side chain was immobilized onto the solid support via the Mitsunobu reaction. Peptides corresponding to centrally truncated analogs of neuropeptide Y were formed. Cyclization was achieved prior to TFA cleavage from the resin. A further example in this category was the synthesis of cyclic thioether peptides12 on HMPB-MBHA (3) resin.

Synthesis of C-Terminally Modified Peptides

The HMPB linker 1 has been successfully utilized in the synthesis of C-terminally modified peptides by use of a dual linker system.13 The linker was coupled to the amino side chain of a resin-bound lysine residue and a peptide built up through the hydroxymethyl group of the aromatic chain of the linker via conventional Fmoc peptide synthesis.

Coupling of the free amino of the peptide to form 4 followed by concomitant palladium (0) mediated deprotection of the two allyl moieties afforded an intermediate that was subsequently cyclized to 5, containing a dual linker system (1). Treatment of the cyclized peptides with 1% TFA in CH2Cl2 resulted in cleavage from the HMPB linker to yield fully protected, inverted resin-bound peptides 6. These could then be either released from the solid-phase using 100% TFA or be modified at the C-terminus followed by TFA cleavage. A variation in using a dual linker system for the preparation of C-terminally modified peptides involved incorporating a coding tag by using a combination of HMPB (80%) and hydroxymethylbenzoic acid (HMBA) (20%) linkers to attach to the side chain of resin-bound lysine. TFA cleavage releases the HMPB-bound peptide into solution leaving the HMBA-bound peptide available to be identified by Edman sequencing.13

Multiple Release Strategies Involving the HMPB Linker

A multiple release protocol was developed (2),14 which involved the attachment of three different linkers, HMPB (1), 4-hydroxymethylphenoxyacetic acid (HMPA), and 4-hydroxymethylphenylacetic acid, onto aminomethyl resin to generate a multifunctionalized resin 7. Fmoc solid-phase peptide synthesis acid on each linker allowed the generation of tripeptides, each requiring varying degrees of TFA for peptide cleavage. HMPB allows the peptide to be liberated using 1% TFA in CH2Cl2 whilst 95% TFA in CH2Cl2 was needed for peptide release from HMPA. This left a peptide sequence linked to the resin bead via a phenylacetamidomethyl (PAM) linkage, to act as a coding strand, which is easily analyzed by either mass spectrometry or Edman sequencing.

Miscellaneous

The HMPB linker has been attached to generation [3.0] polyamidoamino (PAMAM) dendrimers for the solid-phase synthesis of aryl ethers.3 Linker 1 has also been used to evaluate the synthetic potential of new resins. It has been used as a linker both on magnetite impregnated beads15 and on novel poly(styrene-oxyethylene) grafted copolymer resins.16

Related Reagents.

2-Chlorotrityl chloride resin;17 4-[2,4-dimethoxyphenyl-hydroxymethyl]-phenoxy resin (Rink acid resin);18 9-Fmoc-amino-xanthen-3-yloxy resin (Sieber amide resin).19,20


1. Flörsheimer, A.; Riniker, B., In Peptides 1990 ESCOM; Giralt, E.; Andreu, D., Eds.; ESCOM Science Publishers B. V., 1991, pp 131-133.
2. Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D., Tetrahedron Lett. 1989, 30, 1927.
3. Basso, A.; Evans, B.; Pegg, N.; Bradley, M., Tetrahedron Lett. 2000, 41, 3763.
4. Greenlee, M. L.; Laub, J. B.; Balkovec, J. M.; Hammond, M. L.; Hammond, G. G.; Pompliano, D. L.; Epstein-Toney, J. H.; Bioorg. Med. Chem. Lett. 1999, 9, 2549.
5. Sieber, P., Tetrahedron, Lett. 1987, 28, 6147.
6. Cabrele, C.; Langer, M.; Beck-Sickinger, A. G., J. Org. Chem. 1999, 64, 4353.
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8. Sheppard, R. C.; Williams, B. J., Int. J. Pept. Protein Res. 1982, 20, 451.
9. Riniker, B.; Flörsheimer, A.; Fretz, H.; Sieber, P.; Kamber, B., Tetrahedron 1993, 49, 9307.
10. Alsina, J.; Giralt, E.; Albericio, F.; Tetrahedron Lett. 1996, 37, 4195.
11. Dalcol, I.; Rabanal, F.; Ludevid, M.-D.; Albericio, F.; Giralt, E., J. Org. Chem. 1995, 60, 7575.
12. Jones, D. S.; Gamino, C. A.; Randow, M. E.; Victoria, E. J.; Yu, L.; Coutts, S. M., Tetrahedron Lett. 1998, 39, 6107.
13. Davies, M., Bradley, M.; Angew. Chem. Int. Ed. Engl. 1997, 36, 1097.
14. Cardno, M.; Bradley, M., Tetrahedron Lett. 1996, 37, 135.
15. Szymonifka, M. J.; Chapman, K. T., Tetrahedron Lett. 1995, 36, 1597.
16. Gooding, O. W.; Baudart, S.; Deegan, T. L.; Heisler, K.; Labadie, J. W.; Newcomb, W. S.; Porco, J. A., Jr; Van Eirkeren, P., J. Comb. Chem. 1999, 1, 113.
17. Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G., Int. J. Peptide Protein Res. 1991, 37, 513.
18. Rink, H., Tetrahedron Lett. 1987, 28, 3787.
19. Sieber, P., Tetrahedron Lett. 1987, 28, 2107.
20. Chan, W. C.; White, P. D.; Beythien, J.; Steinauer, R., J. Chem. Soc., Chem. Commun. 1995, 589.

Mark Bradley & Stifun Mittoo

University of Southampton, Southampton, UK



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