4-HYDROXYMETHYL-3-NITROBENZOIC ACID 1

4-Hydroxymethyl-3-nitrobenzoic Acid

OHO

NO2

OH

[82379-38-2] C8H7NO5 (MW 197.15)InChI = 1/C8H7NO5/c10-4-6-2-1-5(8(11)12)3-7(6)9(13)14/h1-

3,10H,4H2,(H,11,12)/f/h11HInChIKey = KUOYLJCVVIDGBZ-WXRBYKJCCC

(linker for photolytic cleavage of protected peptides andoligonucleotides from the solid support)

Physical Data: mp 162–166 ◦C.1

Solubility: soluble in DMF and CH2Cl2.Form Supplied in: pale yellow needles, commercially available.Preparative Methods: obtained from the commercially available

4-bromomethyl-3-nitrobenzoic acid by hydrolysis in an aque-ous suspension of KI at reflux for 18 h2 or in a saturated solutionof NaHCO3 at 100 ◦C for 30 min.3

Handling, Storage, and Precaution: easy to handle, can be storedat room temperature. Nevertheless, dark bottles are recom-mended.

Applications in Peptide and Oligonucleotide Syntheses. 4-Hydroxymethyl-3-nitrobenzoic acid (HMBA linker) has been at-tached to an amino base resin (1). This linker-resin can be used forthe incorporation of carboxylic acids and, after further modifica-tion, these can be cleaved by photolysis. Alternatively, the materialcan be converted into a carbonate base resin for the attachmentof hydroxyl groups which after manipulation can be cleaved byphotolysis. In both cases, the corresponding aldehyde derivative 2is formed (eq 1). This strategy has been applied mainly in peptideand oligonucleotide syntheses.

Peptide Synthesis. The HMBA linker has been used as a linkerfor the photolytic cleavage of protected peptides from the solidsupport. This linker was based on o-nitrobenzyl resins describedby Rich and Gurwara,4 who anchored 4-bromomethyl-3-nitro-benzoic acid on an aminomethyl polystyrene resin. The attach-ment of this acid to a BHA-resin gives rise to the material knownas Nbb-resin.5

The use of the bromomethyl derivative usually implies that theC-terminal amino acid is incorporated onto the resin using the-cesium salt method,6 although in some cases the yield of thisreaction is low and poor reproducibility is obtained. The limita-tions inherent in this method are overcome by converting the bro-momethylnitrobenzyl handle to the corresponding hydroxymethylderivative.

Stability. The linkage between the handle and carboxyl groupsis completely stable to Boc/Bzl protocols, but is not fully compat-ible with the Fmoc/tBu strategy. For example, the peptide-linker

bond is not completely stable towards the treatment with piperi-dine, which is used in the removal of Fmoc, and so the synthesisof long peptides using this strategy is not recommended.

O

NO2

OO

O

R

O

NO2

HO

R

R

R

O

NO

O H

R

O

NO2

HO

NH

NHO

NO2

OR

O

2

= Solid supportL= Leaving group

R

NH

NH

RNH

RNHO

NO2

O

O

L

OHO

NO2

HO

R-COOH

Oligomer-OH + CO2 + 2 (1)

Oligomer-COOH + 2

R-OH

1

hν

1

hν

The stability of Nbb-resins to thiolysis has also been demon-strated in a three-dimensional orthogonal scheme. In this case theDts group, which is labile to thiolysis, is used for N α-protectionand tBu is used to protect the side chain functionalities.7

Incorporation of the C-terminal Amino Acid. This can beachieved in two ways:1 (i) first, direct incorporation of the linkeronto the amino resin, followed by anchoring of the first protectedamino acid with DIPCI and DMAP; (ii) second, by esterifying theprotected amino acid with the linker in solution to give a ‘pre-formed handle,’ and its subsequent incorporation onto the aminoresin (eq 2). Although the 3-nitro-HMBA linker can be incorpo-rated into the resin with carbodiimide and other coupling reagents,it has also been incorporated through its trichlorophenyl ester bytwo alternative approaches.3 In the preformed handle, the ester-ification process has two functions: first to protect the carboxylgroup during the reaction with the amino acid and second to acti-vate the ester for its incorporation onto the solid support.

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2 4-HYDROXYMETHYL-3-NITROBENZOIC ACID

R

RR NH2

Cl

Cl

Cl

O

O

O2N

R'HNO

O

R

NH2

R′HNOH

O

R

R = Resin

R = side chain of amino acid

R′ = amino protecting group

DIPCI, DMAP (2)

R NH2

OH

O

O2N

HO

Cl

Cl

Cl

OHDCC

HOBt

O

O

O2N

HO

Cl

Cl

Cl

HOBt

DIPCI, HOBt

NH

O

O2N

HO

R′ΗΝOH

O

R

NH

O

O2NR

R′HNO

O

R

DIPCI, DMAP

Cleavage. (i) By photolysis: The peptide-Nbb-resin is sus-pended in toluene/TFE (4:1), the suspension is degassed under aslight vacuum (water pump) and purged with argon three times.The photolysis is carried out by irradiating the sample at 350–360nm for 9–14 h, while maintaining the vigorous magnetic stir-ring. The resin is filtered off and washed with toluene/TFE (1:4),DCM, and MeOH. The combined filtrates are evaporated to dry-ness. These conditions give cleavage yields up to 85% and highpurities are obtained. A number of solvent systems were stud-ied before the one described above was determined to be opti-mal for the photolytic cleavage of the peptides from the resin.It was found that the yield obtained was dependent on the de-gree of swelling of the polystyrene, and the use of 20% of TFE8

in the solvent mixture was critical. One limitation of the cleav-age step is the amount of peptide-resin to be cleaved. Only rel-atively small quantities of the resin may be photolyzed at onetime (i.e., up to 500 mg). Azo and azoxy compounds can be pro-duced from the o-nitrosobenzaldehyde9 by product formed dur-ing the photolysis reactions. These compounds have a deep redcolor and act as an internal light filters, thus reducing the cleavageyields.

(ii) By Nucleophilic Displacement: Different carboxyl-terminalderivatives of peptides such as carboxylic acids, alkyl amides,and alkyl esters can be obtained using nucleophilic displacementmethod.10 This option is possible due to the electron-withdrawingproperties of both the carboxamide group and the nitro group atthe para and ortho positions, respectively, to the benzylic linkage.This method has given good yields for methyl esters and methylamides. The use of more hindered amines leads to a considerablereduction in the yields.3

Formation of Diketopiperazines (DKP). Coupling of the thirdamino acid must be carried out carefully when this linker is usedbecause the level of DKP formation11 is generally very high. Thisreaction takes place once the amino protecting group of the sec-ond amino acid has been removed and it is base-catalyzed. Theside reaction is therefore most prevelant in an Fmoc/tBu strategy.When the Boc/Bzl protocol is used, the formation of DKP canbe avoided by employing an in situ neutralization coupling af-ter acidolytic removal of the Boc group with DCC12 or BOP13

as the coupling agent. In the Fmoc/tBu strategy, there are twoways to avoid DKP formation: (i) first, incorporation of the pro-tected dipeptide corresponding to the second and third amino acidsof the sequence,14 and (ii) second, incorporation of the secondamino acid with Trt as the amino protecting group,15 removalof the Trt group, and in situ neutralization coupling as outlinedabove.

Applications.

Cyclic Dipeptides (DKP). These systems have been synthe-sized by exploiting the side reaction mentioned above. A studyof the cyclization conditions indicated that the best procedure in-volves base catalysis (0.3 M DIEA). By using this methodology,racemization cannot be detected.16

Conversion to an Active Carbonate Resin for Anchoring ofthe Hydroxyl Function. The conversion of the resin into itsmixed succinimidyl carbonate (an active carbonate) by reac-tion with DSC allows the incorporation of molecules througha hydroxyl function [Boc-Threoninol(Bzl)-OH, Boc-Ser-OAll,

A list of General Abbreviations appears on the front Endpapers

4-HYDROXYMETHYL-3-NITROBENZOIC ACID 3

Boc-Tyr-OAll]. Peptide synthesis in the C→N direction, accord-ing to a Boc/Bzl protocol, can then be performed. When elonga-tion of the peptide chain is complete, photolysis renders the freehydroxy peptide.17

Oligonucleotide Synthesis. 5′-O-Dimethoxytrityl-3′-O-succinatothymidine has been coupled in solution to thetrichlorophenyl ester of 4-hydroxymethyl-3-nitrobenzoic acid.The resulting activated ester has been attached to LCAA-CPG,the usual solid support used in oligonucleotide synthesis. Thelinker-support bond is compatible with the standard automatedphosphoramidite oligonucleotide synthesis.18

The cleavage of the oligonucleotide from the solid support canbe achieved by photolysis to give oligonucleotide 3′-succinates inyields comparable to those obtained by standard alkaline hydro-lysis, where the product is the 3′-hydroxy oligonucleotide. Theuse of phosphoramidites containing allyloxy protecting groupsthat are labile to Pd0 allows the deprotection of oligonucleotideswithout the use of a base. The ability to carry out the cleavagereaction before or after the deprotection of nucleobases and phos-photriesters qualifies this support as an orthogonal linker.19

As described above for the peptide synthesis, chloroformatescan be formed with this system. This compound is used to formcarbonate bonds and, in this way, free 3′-hydroxyl and othermodified oligonucleotides containing carboxylic acids at the 3′end have been synthesized.20

Related Reagents. 4-(4-Hydroxymethyl-2-methoxy-5-nitro-phenoxy)butanoic Acid; 4-[4-(1-Hydroxyethyl)-2-methoxy-5-nitrophenoxy]butanoic Acid.21

1. Lloyd-Williams, P.; Gairí, M., Albericio, F.; Giralt, E., Tetrahedron 1991,47, 9867.

2. Kneib-Cordonier, N.; Albericio, F.; Barany, G., Int. J. Peptide ProteinRes. 1990, 35, 527.

3. Nicolás, E.; Clemente, J.; Albericio, F.; Pedroso, E.; Giralt, E.,Tetrahedron Lett. 1992, 33, 2183.

4. Rich, D. H.; Gurwara, S. K., J. Am. Chem. Soc 1975, 97, 1575.

5. Giralt, E.; Albericio, F.; Andreu, D.; Eritja, R.; Martin, P.; Pedroso, E.,An. Quím. 1981, 77, 120.

6. Gisin, B. F., Helv. Chim. Acta. 1973, 56, 1476.

7. Barany, G.; Albericio, F., J. Am. Chem. Soc. 1985, 107, 4936.

8. Giralt, E.; Eritja, R.; Pedroso, E.; Granier, C.; van Rietschoten, J.,Tetrahedron 1986, 42, 691.

9. Patchornik, A.; Amit, B.; Woodward, R. B., J. Am. Chem. Soc. 1970, 92,6333.

10. (a) Albericio, F.; Ripoll, R.; Pedroso, E.; Giralt, E., Afinidad 1985, 42,491. (b) Giralt, E.; Celma, C.; Ludevid, M. D.; Pedroso, E., Int. J. PeptideProtein Res. 1987, 29, 647. (c) Nicolás, E.; Clemente, J.; Ferrer, T.;Albericio, F.; Giralt, E., Tetrahedron 1997, 53, 3179.

11. Giralt, E.; Eritja, R.; Pedroso, E., Tetrahedron Lett. 1981, 22, 3779.

12. Suzuki, K.; Nitta, K.; Endo, N., Chem. Pharm. Bull. 1975, 23, 222.

13. Gairí, M.; Lloyd-Williams, P.; Albericio, F.; Giralt, E., Tetrahedron Lett.1990, 31, 7363.

14. Pedroso, E.; Grandas, A.; de Las Heras, X.; Eritja, R.; Giralt, E.,Tetrahedron Lett. 1986, 27, 743.

15. Alsina, J.; Giralt, E.; Albericio, F., Tetrahedron Lett. 1996, 37, 4195.

16. Giralt, E.; Eritja, R.; Josa, J.; Kuklinski, C.; Pedroso, E., Synthesis 1985,181.

17. Alsina, J.; Chiva, C.; Ortiz, M.; Rabanal, F.; Giralt, E.; Albericio, F.,Tetrahedron Lett. 1997, 38, 883.

18. Greenberg, M. M., Tetrahedron Lett. 1993, 34, 251.

19. Greenberg, M. M.; Gilmore, J. L., J. Org. Chem. 1994, 59, 746.

20. (a) Greenberg, M. M., Tetrahedron 1995, 51, 29. (b) Yoo,D. J.; Greenberg, M. M., J. Org. Chem. 1995, 60, 3358. (c) Venkatesan,H.; Greenberg, M. M., J. Org. Chem. 1996, 61, 525. (d) McMinn, D. L.;Greenberg, M. M., Tetrahedron 1996, 52, 3827.

21. Holmes, C. P.; Jones, D. G., J. Org. Chem. 1995, 60, 2318.

Beatriz G. de la TorreInstitut de Biologia Molecular de Barcelona,

Barcelona, Spain

Ramon EritjaBarcelona Biomedical Research Institute,

Barcelona, Spain

Fernando AlbericioUniversity of Barcelona, Barcelona, Spain

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