New phenylfluorenyl based linkers for solid phase synthesis

Article abstract of DOI:10.1016/S0040-4039(00)01654-3

The synthesis of 9-(4-hydroxyphenyl)-9-H-fluoren-9-ol and 5-(4-(9-hydroxy-9H-fluoren-9-yl)-phenoxy)-pentanoic acid and their applications as new linkers for solid phase synthesis is reported. These supports show higher acid stability compared to standard trityl resins. Carboxylic acids and amines are immobilized and cleaved off after further modifications. TFA treatment releases the products in high yield and excellent purity. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01654-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 9037–9042
New phenylfluorenyl based linkers for solid phase synthesis
Konrad H. Bleicher,* Christian Lutz and Yves Wu¨thrich
F. Hoffmann-La Roche AG, Pharma Research, CH-4070 Basel, Switzerland
Received 30 August 2000; accepted 21 September 2000
Abstract
The synthesis of 9-(4-hydroxyphenyl)-9-H-fluoren-9-ol and 5-(4-(9-hydroxy-9H-fluoren-9-yl)-phenoxy)-
pentanoic acid and their applications as new linkers for solid phase synthesis is reported. These supports
show higher acid stability compared to standard trityl resins. Carboxylic acids and amines are immobi-
lized and cleaved off after further modifications. TFA treatment releases the products in high yield and
excellent purity. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: linkers; solid phase synthesis; peptide synthesis; inverse peptide synthesis; 2-aminothiazoles.
Solid phase organic synthesis (SPOS) has become a useful tool for the generation of diverse
compound libraries in the drug discovery and lead optimization process.1 In recent publications
an increasing number of solution phase methodologies were transferred successfully to solid
phase synthesis.2 One of the key issues in SPOS is to find a proper linker system for the
attachment of the first building block to the solid support. Trityl resins were described for the
immobilization of various nucleophiles like carboxylic acids,3 alcohols4 and amines.5 The
convenient loading of amino acids and the mild cleaving conditions make these resins very
attractive for the syntheses of peptides and protected peptide fragments.3,6 For solid phase
synthesis of small molecules the acid sensitivity of trityl resins often turns out to be a drawback,
therefore they can only be used for a limited range of reactions. The 9-phenylfluoren-9-yl group
(PhFl) has been described for the protection of primary and secondary amines7 as a more acid
stable alternative to the trityl group. This protecting group shows a 6000 times higher stability
than trityl due to the antiaromatic character of the fluorenyl cation produced upon acidic
treatment.8 We have investigated the phenylfluorenyl protecting strategy as a linkage approach
in SPOS for several nucleophiles as disclosed in earlier publications.9 Henkel et al. describe an
analogous approach for the immobilization of amino acids10 and recently a publication
appeared using the PhFl linker concept for soluble polymer supported chemistry.11
* Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01654-3

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Herein we wish to report new phenylfluorenyl based linkers as interesting alternatives to the
trityl- and PhFl-modified resins published so far. The syntheses of two derivatives are described
that can be used for the modification of either chloromethyl polystyrene or any aminomethyl
modified resins12 (Scheme 1).
Scheme 1. (i) n-BuLi, THF −78°C; (ii) 9-fluorenone −78°C to rt; (iii) NH4Cl sat.; (iv) For R=TIPS: TBAF (1M) in
THF; for R=allyl: 10 mol% Pd(PPh3)4, morpholine (5 equiv.); (v) ethyl 5-bromovalerate (5), K2CO3, acetone, reflux
Starting from either allyl- or triisopropylsilyl protected 4-bromophenol 1 protected phenylflu-
orenyl alcohols 2, 3 are obtained in good yield after lithiation and 9-fluorenone quenching.
Subsequent deprotection with either TBAF or Pd(PPh3)4/morpholine13 results in deprotected
alcohol 4 in excellent yields. Compound 6 is obtained by ether formation of 4 with ethyl
5-bromovalerate 5 followed by ester hydrolysis. Alternatively either 9-(4-hydroxyphenyl)-9H-
fluoren-9-ol 4 or 5-(4-(9-hydroxy-9H-fluoren-9-yl)-phenoxy)-pentanoic acid 6 are linked to
Merrifield resin and 1% cross linked aminomethyl polystyrene, respectively (Scheme 2).14
Scheme 2. (i) CsꢀSalt of 4, DMF; (ii) 20% AcCl, DCM; (iii) 6, TBTU, NMM, DMF
To investigate the acid stability of the oxygen-modified PhFl-linkage resin, 10 was loaded with
FmocꢀPheꢀOH15 and treated under several acidic conditions. FmocꢀPheꢀmodified TCP16 and
2-chlorotrityl6 resins were also used for direct comparison. After 30 min the resins were filtered
off and the solutions were evaporated and the white solids obtained were dried, weighed and
analyzed by HPLC. Scheme 3 shows the amount of recovered FmocꢀPheꢀOH as a percentage
of the theoretical total recovery.

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Scheme 3. Comparison of linker stability. The resins were treated under several acidic conditions for 30 min. (1) acetic
acid/DCM (1:9); (2) acetic acid/DCM (2:8); (3) TFA/MeOH/DCM (1:10:89); (4) acetic acid/2,2,2-trifluoroethanol/
DCM (1:1:8)
While carboxylic acids are cleaved quantitatively from trityl resins under very mild conditions
(dilute AcOH or hexafluoroisopropanol)6 less than 5% cleavage is observed for the PhFl
derivative after treatment with 10% AcOH for 30 min. This resin shows reasonable stability up
to 1% TFA. Quantitative cleavage of the compound is obtained by treatment with 10% TFA for
60 min (data not shown). To demonstrate the applicability of the resin for peptide synthesis a
model compound (FmocꢀAlaꢀValꢀPheꢀOH) was synthesized using standard conditions.17 The
tripeptide was obtained with a crude yield of 83% and an HPLC purity of >95%.
To show the utility of these resins for the immobilization of N-nucleophiles an ‘inverse’
peptide synthesis was performed on both resins (8 and 10). Loading of phenylalanine allyl ester
via the N-terminus, allyl ester cleavage and coupling of phenylalanine allyl ester gave the
corresponding dipeptide ester.18 The product 13 was obtained in good yield and high purity
(Scheme 4).
Scheme 4. (i) Pd(PPh3)4, morpholine; (ii) phenylalanine allyl ester, TBTU, DIPEA; (iii) 20% TFA, 2% Et3SiH
For the original PhFl polystyrene resin9 the linkage of more basic amines turned out to be too
stable for reasonable product recovery after TFA treatment. To investigate the cleavage
conditions for such basic amines from the oxygen modified PhFl resins described herein
9-(4-methoxyphenyl)-9-H-fluoren-9-ol was synthesized in analogy to compounds 2 and 3
(Scheme 1 R=CH3). Conversion of the alcohol to the chloride and addition of benzylamine
resulted in a model compound that allowed us to investigate the cleavage conditions for the

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CꢀN bond in solution. Therefore the compound was treated under various acidic conditions and
the cleavage rate was followed by HPLC analysis. A quantitative cleavage was observed with
40% TFA, 10% Et3SiH in DCM/MeOH (9:1) for 90 min. A decrease of the scavenging reagent
led to an increase in reaction time. In our hands the most reasonable conditions for a solid
phase approach turned out to be a 4 h treatment with 50% TFA, 2% Et3SiH in DCM/MeOH
(9:1).
As an example for the immobilization and recovery of basic amines after a multi step reaction
the synthesis of compound 17 (Scheme 5)19 was carried out. Immobilization of 3-nitrobenzyl-
amine followed by SnCl2 reduction gave resin 14. Thiourea 15 was cleanly formed by reaction
with FmocNCS. Fmoc deprotection and cyclisation with a-bromo acetophenone resulted in the
formation of resin bound 2-aminothiazole 16.
Scheme 5. (i) 3-Nitrobenzylamine; (ii) SnCl2; (iii) FmocNCS; (iv) 20% piperidine/DMF; (v) a-bromoacetophenone,
(vi) 50% TFA, 2% Et3SiH in DCM/MeOH (9:1)
The cleavage of the benzylamine moiety from the resin was investigated under various
conditions. Et3SiH as a scavenger turned out to be essential for the final cleavage step. Omission
of triethylsilane resulted in decomposition of the products. Additionally for resin 8 a liberation
of the linker moiety was observed. Too high concentrations of TFA tend to cleave the benzyl
ether bond resulting in the cleavage of the linker moiety from the solid support. Similar side
reactions have been reported for other linker systems fixed via a benzyl ether bond.20 This side
reaction is prevented when using resin 10. Here the product is formed with a HPLC purity of
>95% and a crude yield of 70% after six steps using a cleavage cocktail of 50% TFA, 2% Et3SiH
in DCM/MeOH (9:1) for 4 h.21 Since resin 8 is not stable under these cleavage conditions, resin
10 is recommended when higher TFA concentrations are needed for releasing the product from
the solid support.
In conclusion, two new phenylfluorenyl based linkers for solid phase organic synthesis are
introduced and their acid stability is compared to known trityl linker systems. We have
demonstrated that oxygen and nitrogen nucleophiles can be immobilized, modified and cleaved
off in good yield and high purity under appropriate cleavage conditions.
References
1. (a) Cowley, P. M.; Rees, D. C. Cur. Med. Chem. 1997, 4, 211–227; (b) Gordon, E. M.; Barrett, R. W.; Dower,
W. J.; Fodor, S. P. A.; Gallop, M. A. J. Med. Chem. 1994, 37, 1385–1401; (c) Gallop, M. A.; Barrett, R. W.;
Dower, W. J.; Fodor, S. P. A.; Gordon, E. M J. Med. Chem. 1994, 37, 1233–1251.

9041
2. (a) Booth, S.; Hermkens, P. H. H.; Ottenheijm, H. C. J.; Rees, D. C. Tetrahedron 1998, 54, 15385–15441; (b)
Hermkens, P. H. H.; Ottenheijm, H. C. J.; Rees, D. C. Tetrahedron 1997, 53, 5643–5678; (c) Hermkens, P. H. H.;
Ottenheijm, H. C. J.; Rees, D. C. Tetrahedron 1996, 52, 4527–4554.
3. Barlos, K.; Gatos, D.; Kallitsis, J.; Papaphotiu, G.; Sotiriu, P.; Wenqing, Y.; Scha¨fer, W. Tetrahedron Lett. 1989,
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4. Borhan, B.; Wilson, J. A.; Gasch, M. J.; Ko, Y.; Kurth, D. M.; Kurth, M. J. J. Org. Chem. 1995, 60, 7375–7378.
5. Hoekstra, W. J.; Greco, M. N.; Yabut, S. C.; Hulshizer, B. L.; Maryanoff, B. E. Tetrahedron Lett. 1997, 38,
2629–2632.
6. Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. Int. J. Peptide Protein Res. 1991, 37, 513–520.
7. (a) Gerspacher, M.; Rapoport, H. J. Org. Chem. 1991, 56, 3700–3706; (b) Gmeiner, P.; Feldman, P. L.;
Chu-Moyer, M. Y.; Rapoport, H. J. Org. Chem. 1990, 55, 3068–3074; (c) Bradley, D. C.; Rapoport, H. J. Org.
Chem. 1985, 50, 1239–1246.
8. Bolton, R.; Chapman, N. B.; Shorter, J. J. Chem. Soc. 1964, 1895–1906.
9. (a) Bleicher, K. H.; Wareing, J. R. Tetrahedron Lett. 1998, 39, 4587–4590; (b) Bleicher, K. H.; Wareing, J. R.
Tetrahedron Lett. 1998, 39, 4591–4594.
10. Henkel, B.; Bayer, E. Tetrahedron Lett. 1998, 39, 9401–9402.
11. Gosselin, F.; Van Betsbrugge, J.; Hatam, M.; Lubell, W. D. J. Org. Chem. 1999, 64, 2486–2493.
12. (a) Synthesis of 4: A three neck flask with argon inlet, low temperature thermometer, stirring bar and septum was
charged with 1 (either allyl or TIPS protected, 1.0 equiv., 1.5 M solution in THF). n-Butyllithium (1.2 equiv., 1.6
M solution in hexane) was added at −78°C. After 20 min a solution of 9-fluorenone (1.2 equiv. in THF) was
added slowly and warmed to rt. After hydrolysis (NH4Cl sat.), TBME extraction and evaporation of the solvent
the crude product was purified by Kieselgel chromatography using ethyl acetate/hexane. The products (2, 3) were
obtained in 93 and 74% yield, respectively. Compound 2 was deprotected with TBAF (1.2 equiv. in THF) at rt
overnight. Compound 3 was treated with Pd(PPh3)4/morpholine as described.13 (b) Synthesis of 6: A round
bottomed flask with reflux condenser was charged with K2CO3 (5 equiv.), ethyl-5-bromovalerate (2 equiv.),
sodium iodide (cat.) and 4 (1.0 equiv.) in acetone. The mixture was refluxed for 8 h, filtered and evaporated. The
crude product was dissolved in dioxan, conc. NaOH (9:1) and warmed to 100°C for 2 h. After acidification (20%
HCl solution, pH 1) the precipitated product was filtered off and dried under vacuum. 5-(4-(9-Hydroxy-9H-
fluoren-9-yl)-phenoxy)-pentanoic acid (6) was obtained in 75% yield.
13. Eicher, T.; Ott, M.; Speicher, A. Synthesis 1996, 755–762.
14. (a) Coupling of 4 to Merrifield resin: 1.2 equiv. 4 (Csꢀsalt) and cat. KI were dissolved in DMF. Merrifield resin
(0.7 mequiv./g) was added and the slurry was shaken for 2 days at 80°C and extensively washed with DMF/H2O,
MeOH, THF, DCM. A loading of 0.61 mequiv./g was obtained as determined by weight increase. (b) Coupling
of 6 to aminomethyl polystyrene: 1.5 equiv. of 6, 1.5 equiv. TBTU, 1.5 equiv. NMM were added in DMF to
aminomethyl polystyrene (1% DVB, 1.19 mequiv./g) and the slurry was shaken for 16 h. After washing with
DMF, MeOH, DCM (×2) Kaiser test showed 100% conversion resulting in a loading of 0.84 mequiv./g. (c)
Activation of resins 8 and 10: The resins were treated with 20% acetyl chloride in DCM for 6 h. The resins were
washed with DCM and dried under vacuum.
15. Loading of FmocꢀPheꢀOH: The resin was treated with 2 equiv. FmocꢀPheꢀOH, 4 equiv. DIPEA in DCM for 2
h. The reaction was quenched with MeOH and washed with DMF, MeOH, DCM (×2). A quantitative loading
was obtained as determined by weight increase and elemental analysis.
16. Bayer, E.; Goldammer, C. German Pat. DE 43 06 839 A1 1994; Bayer, E.; Henkel, B.; Zhang, L.; Clausen, N.;
Goldammer, C.; Panhaus, G. Peptides 1994, Proceedings of the 23rd European Peptide Symposium, Maia, H. L.
S., Ed.; Escom: Leiden, 1995, pp. 201–202. The TCP resin is commercially available from PepChem (Tu¨bingen,
Germany).
17. 100 mg of the resin obtained (see Ref. 15) was treated with 20% piperidine/DMF for 30 min for Fmoc
deprotection. The resin was washed with DMF, DCM (×2). 2 equiv. of FmocꢀVal, TBTU and DIPEA in DMF
were added. After 1 h the resin was washed with DMF, MeOH, DCM (×2). Fmoc cleavage and coupling of
FmocꢀAla were performed as described above. The tripeptide was cleaved with 10% TFA, 2% Et3SiH in
DCM/MeOH (9:1) for 1 h. The solution was evaporated and the white solid obtained dried under vacuum.
18. (a) Loading of phenylalanine allyl ester: 2 equiv. of phenylalanine allyl ester toluenesulfonate were dissolved in
DCM by adding 4 equiv. DIPEA. The solution was added to the resin and stirred for 2 h at rt. The reaction was
quenched with MeOH and the resin washed with DMF, MeOH, THF, DCM (×2). A quantitative loading was
obtained as determined by elemental analysis. (b) Allyl ester cleavage: 0.2 equiv. Pd(PPh3)4 and 10 equiv.

9042
morpholine were dissolved in DCM and added to the resin. After 1 h the resin was rinsed with DCM, a solution
of sodium diethyl dithiocarbamate/DIPEA (1:1) in DMF (20 mmol), DMF, MeOH, and DCM (×2). (c) Coupling
of phenylalanine allyl ester: 2 equiv. of phenylalanine allyl ester toluenesulfonate, 2 equiv. TBTU, 2 equiv. DIPEA
in DMF were added. After 1 h the resin was washed with DMF, MeOH, DCM (×2). The compound was cleaved
with 20% TFA, 2% Et3SiH in DCM/MeOH (9:1) for 1 h. The cleavage procedure was repeated, the solutions
combined and evaporated. The product was dried under vacuum overnight and obtained as a white solid.
19. Kearney, P. C.; Fernandez, M.; Flygare, J. A. J. Org. Chem. 1998, 63, 196–200.
20. Novabiochem Catalog & Peptide Synthesis Handbook; ‘Synthesis Notes’, 1997/98; p. 49.
21. (a) Loading of nitrobenzylamine: Resin 10 was treated with 3-nitrobenzylamine×HCl (2.0 equiv.) and DIPEA (4.0
equiv.) in DCM/DMF (9:1). After 2 h the reaction was quenched with MeOH and the resin washed with DMF,
MeOH, DCM (×2). A loading of 87% was obtained as determined by elemental analysis. No further optimization
for increase of the loading (excess of reagent or increase of reaction time etc.) was undertaken. (b) SnCl2-reduc-
tion: The resin was suspended in DMF and 5 equiv. SnCl2×2 H2O were added. After 16 h the resin was washed
with a solution of sodium diethyl dithiocarbamate/DIPEA (1:1) in DMF (20 mmol), DMF, MeOH, DCM (×2).
(c) Urea formation and deprotection: FmocNCS (1.5 equiv.) in DCM was added for 4 h. After washing with DMF
and DCM (×2) Fmoc was cleaved with piperidine (20% in DMF) for 30 min. (d) Thiazole formation: The
a-bromoacetophenone (3 equiv.) was dissolved in dioxan and added to the resin. After 3 h the resin was washed
with DMF, MeOH, DCM (×2) and the product cleaved off with 50% TFA, 2% Et3SiH in DCM/MeOH (9:1) for
4 h.
.