p-hydroxymethylbenzoic acid

[3006-96-0]  · C8H8O3  · (MW 152.08)

(reagent used as a linker in solid-phase peptide synthesis (SPPS) and solid-phase organic synthesis (SPOS) primarily to anchor carboxylic acids to polymer supports)

Physical Data: mp 183-185 °C.

Solubility: soluble in H2O and most organic solvents.

Form Supplied in: white solid, widely commercially available.

Purification: crystallizes from cold H2O, colorless needles.1-3

Use of the HMBA linker 2 in solid-phase synthetic applications usually starts with commercially available 4-hydroxymethylbenzoic acid, HOCH2C6H4CO2H. 4-hydroxybenzoic acid and its derivatives are coupled to various amine functionalized polymers by DIC activation of the benzoic acid without protection of the hydroxymethyl group as in 1.4,5 The hydroxymethyl group is linked in the next step to a carboxylic acid by esterification. This procedure can be reversed by linking the desired starting material (e.g. 3) to 1 and then linking the resulting conjugate molecule to the solid-phase.6 A less popular protocol involves substitution of bromide in resin-bound 4-bromomethylbenzoate by an alkanoate nucleophile.7

4-Hydroxymethylbenzoic acid is used to modify amine-bearing resins. The difference in the reactivity of amides versus esters is the basis of the ability of the HMBA linker to be cleaved cleanly from the target molecules after synthetic operations are complete. Synthetic steps before the cleavage step result in the elaboration of 5.

Linker/resin assemblies in solid-phase synthesis function similarly to protecting groups in SPOS.8-11 Thus the moiety linking the desired molecule to the resin needs to survive conditions used to elaborate or elongate the desired molecule. Furthermore, the linker/resin assembly needs to be cleaved efficiently from the desired molecule once synthetic manipulations are complete. HMBA linker 1 (1) usually fulfills the above requirements by availing the difference in chemical stabilities between the amide moiety in 2 and the ester in 4. After the desired series of solid-phase chemical steps are performed the more stable amide unit remains bound to the solid-phase while the ester is cleaved from the solid-phase under either basic, highly acidic or photolytic conditions to yield the desired material 5.

Applications of the HMBA Linker to Solid-Phase Peptide Synthesis

In SPPS there are at least two ways in which the growing chain in 2 can be compromised. (1) Stereogenic carbon atoms next to the ester may be racemized. This is the first problem which investigators who developed new SPPS techniques explored.12 Stereochemical integrity of the a-carbon in HMBA-linked peptides appears to be sound since 1 has been used as a linker quite often in SPPS. (2) A free amine on the terminus may cleave the growing chain from the solid-phase by formation of an amide.

Intramolecular aminolysis of the ester by the amino terminus, (7) appears to be problematic with the HMBA linker only at the dipeptide stage of SPPS; under these conditions d-lactams slowly evolve (2).13 Thus, synthetic operations should follow amine deprotection as soon as possible.

The 3-nitro derivative of the HMBA linker, 4-hydroxymethyl-3-nitrobenzoic acid was first developed to facilitate the cleavage of the HMBA ester anchorage from the solid-phase under conditions of aminolysis (e.g. cleavage step resulting in Z=NHR in 1) and photolysis.14-18 In exploratory work, facile aminolysis of 2-nitro-4-oxymethylphenyl resin was demonstrated in SPPS with a modified styrene resin.19 Currently, aminobenzhydryl resins are covalently attached to the 3-nitroHMBA linker by an amide bond. After the desired elongation of the peptide chain is complete, photolysis of ester-bound 3-nitroHMBA results in peptides with free carboxylic acid termini.7,20 Aminolysis of the HMBA esters is routinely performed to yield peptides with primary amides at the C-termini.6,7,21-23 Treatment of 3-nitroHMBA modified resins with ammonia in 2-propanol results in efficient cleavage from the solid-phase to yield peptide amides. When primary amines are used in the aminolysis protocol, N-substituted amides are obtained.7 When the cleavage step is run in MeOH instead of i-PrOH (see 1), methyl esters result due to the decreased steric demand of MeOH.24

Solid-Phase Synthetic Organic Applications of the HMBA Linker

Schemes for SPOS employed to attach and cleave the HMBA linker/resin assembly from the desired material are necessarily more diverse than those of SPPS due to increased diversity of the synthetic targets. SPPS involves iterative, nearly identical operations geared toward elongation of a peptide whereas SPOS more often involves smaller molecules reached by multiple, diverse steps. The stability of HMBA towards a wide variety of chemistry, especially acid, often renders the HMBA linker ideal for SPOS.

The HMBA linker can survive hydrofluoric acid3 and trifluoroacetic acid, but cleaves from the solid-phase upon treatment with triflic acid.2 This point was exemplified in the application of the HMBA linker to the acidic conditions needed for Fisher indole synthesis. After the synthetic protocol was complete, the desired material was cleaved from the resin under basic conditions (MeOH/Et3N 9:1, 50 °C).25 A related TFA-catalyzed iminium cyclization has been used in the solid-phase synthesis of spiroindolines (9 in 3).26 The relative inertness of HMBA to acid and high temperature made it possible to link aryl allyl ether functionality to mesoporous molecular sieves and silica-based supports to explore these polymers as solid-phase supports for Claisen rearrangements at temperatures between 180 and 200 °C, (10 and 11 in 3).27 This particular solid-phase was chosen because most polymeric supports (including polystyrene) have glass transition temperatures below 150 °C. The hardy nature of the HMBA linker allowed application of continuous cellulose membranes to parallel SPOS in the synthesis of resin-bound peptides, peptoids, and carbohydrate-peptide conjugates.28 Since the HMBA linker is stable in the presence of a wide range of reagents, it is a good choice when faced with the need to orthogonally protect two functional groups on the solid phase, as in the case of the two primary amines in the synthesis of peptide conjugates of spermidine.2 Similar perspectives as the preceding one utilize the HMBA linker in the development of SPOS protocols for biomimetic oligosulfones, oligosulfoxides, and oligothioethers.29

The HMBA linker has been successfully applied to the parallel solid-phase synthesis of derivatives of the antifungal ether miconazole.30,31 Complex linkers incorporating ether functionality (as ‘safety catch linkers’) also include HMBA.30 The feasibility of a Mitsunobu C-C bond formation has been explored on the solid-phase with the HMBA linker. N,N,NŽ,NŽ-tetramethylazodicarboxamide (TMAD) and PBu3 and activated XCH2R derivatives can be used to effect C-C bond formation.4 Loading-dependent (equiv g-1 resin) dialkylation competed with the desired monoalkylation.

Enzymatic reactions proceed under mild conditions at neutral pH. An enzyme-labile derivative of the HMBA linker, 3-hydroxymethyl-6-oxyacetyl benzoate in 4, offers greater tolerance of functional groups.5 Upon enzyme-mediated cleavage of the O-acetyl group in 12, the aromatic benzene ring becomes more electron-rich, which leads to solvolysis of the O-carbonyl group holding the synthetic substrate 13 to the solid phase. The compatible functional groups were amino acids, carboxylic acids, and carbohydrates in the form of protected nucleosides.

Abbreviations. DCM: dichloromethane; DIC: N,NŽ-diisopropylcarbodiimide; DMA: dimethylacetamide; DMAP: N,N-dimethyaminopyridine; HMBA: p-hydroxymethybenzoate linker; HOBt: 1-hydroxybenztriazole; SPOS: solid-phase organic synthesis; SPPS: solid-phase peptide synthesis; TFA: trifluoroacetic acid.

Related Reagents.

4-Bromomethyl-3-nitrobenzoic acid linker [55715-03-2];7 4-azidomethylbenzoate ester linker prepared on the solid-phase.32,33


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Arthur Cammers-Goodwin

University of Kentucky, Lexington, KY, USA



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