An improved synthesis of N-[(9-hydroxymethyl)-2-fluorenyl]succinamic acid (HMFS), a versatile handle for the solid-phase synthesis of biomolecules

Article abstract of DOI:10.1081/SCC-100000203

The optimization of a synthetic process for the preparation of HMFS handle, which is used for the solid-phase synthesis of peptides and oligonucleotides, is presented.

Full text of DOI:10.1081/SCC-100000203

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AN IMPROVED SYNTHESIS OF N-[(9-
HYDROXYMETHYL)-2-FLUORENYL]SUCCINAMIC ACID
(HMFS), A VERSATILE HANDLE FOR THE SOLID-PHASE
SYNTHESIS OF BIOMOLECULES
Fernando Albericio a , Montse Cruz a , Laurent Debéthune a , Ramon Eritja a , Ernest
Giralt a , Anna Grandas a , Vicente Marchán a , Jose J. Pastor a , Enrique Pedroso a ,
Francesc Rabanal a & Miriam Royo a
a Department of Organic Chemistry, University of Barcelona, Barcelona, 08028, Spain
Version of record first published: 16 Aug 2006.
To cite this article: Fernando Albericio , Montse Cruz , Laurent Debéthune , Ramon Eritja , Ernest Giralt , Anna Grandas ,
Vicente Marchán , Jose J. Pastor , Enrique Pedroso , Francesc Rabanal & Miriam Royo (2001): AN IMPROVED SYNTHESIS OF
N-[(9-HYDROXYMETHYL)-2-FLUORENYL]SUCCINAMIC ACID (HMFS), A VERSATILE HANDLE FOR THE SOLID-PHASE SYNTHESIS
OF BIOMOLECULES, Synthetic Communications: An International Journal for Rapid Communication of Synthetic Organic
Chemistry, 31:2, 225-232
To link to this article: http://dx.doi.org/10.1081/SCC-100000203
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SYNTHETIC COMMUNICATIONS, 31(2), 225–232 (2001)
AN IMPROVED SYNTHESIS
OF N-[(9-HYDROXYMETHYL)-2-
FLUORENYL]SUCCINAMIC ACID
(HMFS), A VERSATILE HANDLE FOR
THE SOLID-PHASE SYNTHESIS
OF BIOMOLECULES
Fernando Albericio,∗ Montse Cruz, Laurent Debe´thune,
Ramon Eritja, Ernest Giralt, Anna Grandas, Vicente
Marcha´n, Jose J. Pastor, Enrique Pedroso,
Francesc Rabanal, and Miriam Royo
Department of Organic Chemistry, University of Barcelona,
08028-Barcelona, Spain
ABSTRACT
The optimization of a synthetic process for the preparation
of HMFS handle, which is used for the solid-phase synthesis of
peptides and oligonucleotides, is presented.
The preparation of biomolecules, such as peptides or oligonucleotides, and
other organic compounds on a solid matrix is better performed using bifunctional
spacer molecules known as handles or linkers (1). One of the two reactive func-
tionalities of a handle is permanently attached to a suitably functionalized resin,
most often through a stable amide bond, while the growing molecule is temporarily
linked at the other reactive position of the linker.
∗ To whom correspondence should be addressed.
225
Copyright ꢀ 2001 by Marcel Dekker, Inc.
www.dekker.com
C

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ALBERICIO ET AL.
Although the majority of handles relies on acidolysis for the release of the
final molecule from the support, the use of different mechanisms (e.g., photolysis,
fluoridolysis, and base-catalyzed β-elimination) has been exploited for the final
cleavage. Particularly appealing is N-[9-hydroxymethyl)-2-fluorenyl]succinamic
acid (HMFS), which was introduced by our group for the solid-phase synthesis
of protected peptides (2). The finely tuned base-lability of the amino acid-HMFS
bond enables the release of protected peptides from the solid support using a mild
base treatment (piperidine or morpholine—DMF (1 : 4), 0.5–1 h) in high yields and
purities. The linkage between the handle and carboxyl groups is completely stable
to tertiary amines such as N,N-diisopropylethylamine (DIEA) and triethylamine
(TEA), as well as to acidic conditions.
HMFS has been widely used for the solid phase preparation of biological
polymers. In this respect, Sakakibara et al. have recently described the synthesis of
the 238-residue green fluorescent protein (GFP), which is the longest protein ever
chemically synthesized to date. A convergent strategy was used in this work, and
most protected peptides were assembled using Boc/Bzl chemistry on an HMFS-
derivatized resin (3).
Furthermore, the HMFS handle has been used for the synthesis of oligonu-
cleotides (4), 3ꢁ amino-modified oligonucleotides (5), oligonucleotide-peptide hy-
brids (6), and oligonucleopeptides (7), using 1,8-diazobicyclo[5.4.0]-undec-7-ene
(DBU) for the final cleavage rather than ammonia, a reagent commonly used
in oligonucleotides chemistry. Phosphodiester-linked peptide-oligonucleotide hy-
brids have also been assembled on HMFS-functionalized resins, and the target
molecule has been detached from the solid support using either ammonia or tetra-
butylammonium fluoride (8,9).
In our first publication (2), we described a straightforward synthetic route
(Scheme 1) from commercially available 2-aminofluorene. The key step of this
synthetic scheme was the formylation and posterior reduction of 2-(Boc-amino)-
fluorene. Following the experimental procedure outlined in that paper, we and
other researchers have found that 9-hydroxymethyl-2-(Boc-amino)fluorene is
Scheme 1.

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227
obtained occasionally in low yield and with contaminants. The fact that HMFS
is not commercially available, together with the inherent difficulties that may be
encountered in the formylation step, has compelled us to verify the reproducibility
of this potentially troublesome reaction, optimize it, and report a more com-
prehensive and better-detailed protocol. The overall yield may be increased now
to 63%.
Proton NMR interpretation of 9-formyl-2-(Boc-amino)fluorene spectra is
made difficult by the existence of several equilibria, such as keto-enol tautomerism,
restricted rotation of the enol form around C9-C10 bond, and Ccarbamate-N bond.
The 1H-NMR spectrum of the formyl derivative could not be carried out properly
in CDCl3 due to its low solubility. In CD3OD, none of the exchangeable protons
are seen. In addition, it has already been reported that 9-formylfluorene reacts with
methanol to yield the hemiacetal product (10). In DMSO-d6, free of acid and base
impurities, keto-enol tautomerism is slow and exchange is prevented, allowing
the detection of labile protons. However, the urethane proton is shifted downfield
to values similar to those expected for aldehyde protons, making its assignment
difficult. Moreover, water signal probably masks the observation of C9 proton.
Nevertheless, the enol proton could be detected in these conditions (δ 11.1, 11.0).
It appears to be two doublets due to the coupling with the C10 proton and the
presence of two conformers (proportion 6 : 4) generated from restricted rotation
around C9-C10 bond. The enol (δ 11.1, 11.0) proton integrates 0.85 H in agreement
with previous observations reported in the literature for 9-formylfluorene about
the shifting of the equilibrium towards the enol form in polar solvents (11). The
urethane proton also appears as two singulets, possibly arising from restricted
rotation around the Ccarbamate-N bond. Aldehyde proton is temptatively assigned
to δ 9.62, 9.58 (2 s, ca. 0.15 H).
Although aldehydes may be susceptible to air oxidation, in our hands, sam-
ples of 9-formyl-2-(Boc-amino)fluorene kept at 4◦C in regular vials have remained
unchanged for several weeks.
In conclusion, a optimized process for the preparation of HMFS handle,
which is used for the solid-phase synthesis of peptides and oligonucleotides, is
presented. A detailed structural analysis of 9-formyl-2-(Boc-amino)fluorene is
currently being carried out in our lab by 2D-NMR and will be published in due
course.
EXPERIMENTAL
Commercial reagents and solvents are used as received with the following
exceptions. THF is freshly distilled from sodium benzophenone ketyl. 80% NaH
in paraffin oil is used from a recently opened container or after storing it in a des-
iccator under vacuum. Ethyl formate is stored over anhydrous K2CO3 to remove

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ALBERICIO ET AL.
contaminant formic acid. The formylation reaction is performed in a flame-dried
round-bottom flask fit with a rubber septum under a positive Ar pressure. Moisture-
sensitive solutions are transferred via syringe or stainless steel cannula. Analytical
HPLC is carried out on a Shimadzu instrument consisting of two solvent delivery
pumps (model LC-6A), automatic injector (model SIL-6B), variable wavelength
detector (model SPD-6A), system controller (model SCL-6B), and plotter (model
C-R6A). Linear gradient over 30 min of 0.1% TFA in CH3CN and 0.1% aque-
1
ous TFA from 2:8 to 1:0, flow rate 1.0 mL/min, wth UV detection. H-NMR
(200 MHz) spectroscopy is performed on a Varian Gemini 200. Chemical shifts
(δ) are expressed in parts per million downfield from TMS. Coupling constants
are expressed in Hertz. MS of samples are performed on either a Micromass VG
Quattro (FAB-MS) or a Hewlett-Packard HP-5988A (CI) apparatus.
2-(Boc-amino)fluorene
2-Aminofluorene (20 g, 110 mmol) is suspended in a mixture of dioxane-
H2O (2:1) (200 mL) and 2 N NaOH (60 mL) in an ice bath with magnetic stirring.
Boc2O (26.5 g, 121 mmol, 1.1 equiv) is then added and the stirring is continued
at 25◦C. The evolution of the reaction is followed by TLC [Rf = 0.70 for the title
product, CHCl3-MeOH-HOAc (95:5:3)] and the pH is maintained between 9–10
by addition of 2 N NaOH. If, after 24 h, TLC analysis still shows the presence of 2-
aminofluorene [Rf = 0.50, CHCl3–MeOH–HOAc (95:5:3) revealed by ninhydrin],
Boc2O (5 g, 22,8 mmol, 0.21 equiv) is added and the reaction is continued until
the total disappearance of the starting product. The suspension is acidified with
1 M KHSO4 to pH 3. The solid is filtered and washed with cold H2O (50 mL),
dioxane-H2O (2:1) (50 mL), hexane (50 mL), and dried over P2O5. The product,
a pale yellow powder (30.1 g, 97% yield) (12), is shown to be pure by TLC (Rf =
0.70), 1H-NMR [(CDCl3) δ 7.1–7.8 (m, 7 H, Ar), 6.6 (broad s, 1 H, NH urethane),
3.9 (s, 2 H, CH2), 1.6 (s, 9 H, Boc)] and FAB-MS [282.1 (M+H)+, 281 M+].
9-Formyl-2-(Boc-amino)fluorene
A solution of 2-(Boc-amino)fluorene (14.3 g, 51 mmol) in dry THF (70 mL,
freshly distilled from sodium) is carefully added to a suspension of NaH (5 g,
80% in paraffin oil, 167 mmol, 3.3 equiv) (13) in dry THF (20 mL) under Ar
atmosphere. Gas evolution and spontaneous warming are observed (14). Ethyl
formate (10.2 mL, 126 mmol, 2.5 equiv) is then slowly added to avoid vigorous
hydrogenbubbling(15). The initiallythick, lightbrownsuspensionrapidlyclarifies
to a dark brown solution upon addition of ethyl formate. The evolution of the
reaction is followed by TLC [Rf = 0.40 for the title product, CHCl3–MeOH–
HOAc (95:5:3)]. If there is still 2-(Boc-amino)fluorene present after 20 min, more

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229
ethyl formate (4 mL, 50 mmol, 1 equiv) is added and stirring is continued until
the total disappearance of the starting product (16). The reaction is quenched with
ice chips and H2O (100 mL) and the organic solvent is removed in vacuum. The
aqueous solution is washed with diethylether (3 × 50 mL) (17,18), cooled in an
ice bath, and acidified with glacial HOAc until pH 5. The off-white precipitate
that then appears is dissolved in EtOAc (200 mL). The organic phase is washed
with NaHCO3 (4 × 100 mL) and brine (3 × 100 mL) and dried over anhydrous
MgSO4. The solvent is eliminated under reduced pressure, diethylether is added,
and the solution again evaporated. This operation is repeated twice before the
residue (yellow needles), which can be used without further purification, is dried
over P2O5. HPLC, tR 20.5 min. 1H-NMR [(CD3SOCD3), δ 11.1, 11.0 (2 d, 0.85 H,
enol, J = 6.0), 9.62, 9.58 (2 s, 0.15 H, CHO), 9.32, 9.28 (2 s, 1 H, urethane),
7.1–8.3 (m, 8 H, Ar, C10 H enol form), 1.5 (s, 9 H, Boc)].
9-Hydroxymethyl-2-(Boc-amino)fluorene
9-Formyl-2-(Boc-amino)fluorene is suspended in dry methanol (100 mL)
and NaBH4 [2.1 g, 55 mmol, 1.1 equiv with respect to the starting 2-(Boc-
amino)fluorene] is added portionwise. The suspension that rapidly clarifies is
magnetically stirred until the starting product disappears [3–4 h, TLC, Rf = 0.44,
CHCl3–MeOH–HOAc(95:5:3)]. ThereactionmixtureisdilutedwithH2O(300mL)
and acidified with glacial HOAc to pH 5.0, and the precipitate is directly dissolved
in EtOAc (200 mL). The organic phase is washed with NaHCO3 (4 × 100 mL)
and brine (3 × 100 mL), and dried over anhydrous MgSO4. The solvent is ro-
tatory evaporated to give a solid, which can be used without further purification
1
(13.0 g, 82% for this step and the previous one) (19). HPLC, tR 21.9 min. H-
NMR [(CD3OD) δ 7.51 (d, 1 H Ar, J = 2.2 Hz), 7.46 (d, 1 H Ar, J = 6.6 Hz),
7.43 (d, 1 H Ar, J = 8.0 Hz), 7.38 (d, 1 H Ar, J = 7.4 Hz), 7.13 (m, 1 H Ar), 7.11
(m, 1 H Ar), 7.00 (m, 1 H Ar), 3.7–3.8 (m, 1 H, CH), 3.6–3.7 (m, 2 H, CH2), 1.30
(s, 9 H)].
9-Hydroxymethyl-2-aminofluorene
9-Hydroxymethyl-2-(Boc-amino)fluorene (13.0 g, 41.8 mmol) is suspended
in 4 N HCl-dioxane in an ice bath and stirred for 2 h at 0◦C and 1 h at 25◦C. The
solution is concentrated in vacuum to partially eliminate HCl (20). The resulting
solution is chilled and a large excess of CH2Cl2 (800 mL) is added to give a
precipitate, which is filtered, washed thoroughly with CH2Cl2, and dried. The
product is dissolved in the minimum amount of H2O, and the pH is adjusted to 8 by
additionofNaHCO3. Anoilseparates, whichisextractedintoEtOAc(3 × 100mL),
and the combined organic layers are washed with NaHCO3 (4 × 100 mL), brine

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ALBERICIO ET AL.
(3 × 100 mL), and dried over anhydrous MgSO4. After elimination of the solvent,
the residue is dried in vacuum over P2O5. The yellow solid obtained (7.7 g, 87%) is
shown to be homogeneous by TLC [Rf = 0.33, CHCl3–MeOH–HOAc (75:23:2)],
1H-NMR [(CD3OD) δ 7.46 (d, 1 H Ar, J = 7.2 Hz), 7.39 (d, 1 H Ar, J = 7.4 Hz),
7.30 (d, 1 H Ar, J = 7.3 Hz), 7.16 (td, 1 H Ar, J = 7.2, 7.2, 1.0 Hz), 7.03 (td, 1 H
Ar, J = 7.2, 7.2, 1.0 Hz), 6.91 (d, 1 H Ar, J = 2.2 Hz), 6.64 (dd, 1 H Ar, J = 7.6,
2.0 Hz), 3.6–3.9 (m, 3 H CH-CH2)]. FAB-MS [212.7 (M + H)+, 211.7 M+].
N-[(9-Hydroxymethyl)-2-fluorenyl]succinamic
Acid (HMFS Handle)
9-Hydroxymethyl-2-aminofluorene (6.8 g, 32.2 mmol) is dissolved in the
minimum amount of freshly distilled THF (20 mL) and succinic anhydride (3.4 g,
35.4 mmol, 1.1 equiv) is added portion wise over a period of 20 min. A yellowish
solid precipitates spontaneously and the reaction is left for an additional hour or un-
til TLC monitoring indicates the disappearance of starting product. The residue is
filtered and washed with THF–hexane (1:1, 10 mL) and hexane (10 mL). The prod-
uct (9.1 g, 91%) can be used after crystallization from THF–hexane (1:1) with some
drops of MeOH to aid solubilization of the crude. Alternatively, purification can be
achieved by a silica gel dry column chromatography eluting with EtOAc–hexanes
mixtures (from 0:1 to 1:1). TLC [Rf = 0.42, EtOAc–MeOH–HOAc (90:10:3)],
◦
1
mp = 168–169 C. HPLC, tR 12.9 min. H-NMR [(CD3OD) δ 7.65 (d, 1 H Ar,
J = 1.8 Hz), 7.47 (d, 1 H Ar, J = 7.0 Hz), 7.47 (d, 1 H Ar, J = 8.0 Hz), 7.38
(d, 1 H Ar, J = 7.4 Hz), 7.28 (dd, 1 H Ar, J = 8.4, 1.8 Hz), 7.10 (td, 1 H Ar,
J = 7.2, 7.2, 1.0 Hz), 7.01 (td, 1 H Ar, J = 7.2, 6.8, 1.2 Hz), 3.7–3.8 (m, 1 H CH),
3.6–3.7 (m, 2 H, CH2), 2.44 (s, 4 H, CH2–CH2)] and EI-MS [311.6 (M + H)+,
335.5 (M + Na)+, 349.6 (M + K)+].
ACKNOWLEDGMENTS
This work was partially supported by the DGICYT (PB97-0941-C02-01
and PB96-1490) and the Generalitat de Catalunya [Grups Consolidats (1999SGR
00042 and 1998SGR-1) and Centre de Refere`ncia en Biotecnologia].
REFERENCES AND NOTES
1. Songster, M.F.; Barany, G. In Methods in Enzymology, Solid-Phase Peptide
Synthesis; Fields, G.B. Ed.; Academic Press: Orlando, Florida, 1997; Vol.
289, 126–174.

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2. Rabanal, F.; Giralt, E.; Albericio, F. Tetrahedron 1995, 51, 1449.
231
3. Nishiuchi, Y.; Inui, T.; Nishio, H.; Bodi, J.; Kimura, T.; Tsuji, F.I.; Sakakibara,
S. Proc. Natl. Acad. Sci. USA 1998, 95, 13549.
4. Avin˜o, A.; Gu¨imil-Garc´ıa, R.; Diaz, A.; Albericio, F.; Eritja, R. Nucleos.
Nucleot. 1996, 15, 1871.
5. Avin˜o, A.; Gu¨imil-Garc´ıa, R.; Albericio, F.; Mann, M.; Wilm, M.; Neubauer,
G.; Eritja, R. Bioorgan. Med. Chem. 1996, 4, 1649.
6. de la Torre, B.G.; Albericio, F.; Saison-Behmoaras, E.; Bachi, A.; Eritja, R.
Bioconjugate Chem. 1999, 10, 1005–1012.
7. Robles, J.; Maseda, M.; Beltra´n, M.; Concernau, M.; Pedroso, E.; Grandas,
A. Bioconjugate Chem. 1997, 8, 785–788.
8. Beltra´n, M.; Maseda, M.; Robles, J.; Pedroso, E.; Grandas, A. Lett. Pept. Sci.
1997, 4, 147.
9. Robles, J.; Beltra´n, M.; Marcha´n, V.; Pe´rez, Y.; Travesset, I.; Pedroso, E.;
Grandas, A. Tetrahedron 1999, 55, 13251.
10. Carpino, L.A.; Chao, H.G.; Tien, J.H. J. Org. Chem. 1989, 54, 4302.
11. Harcourt, M.P.; More O’Ferrall, R.A. J. Chem. Soc. Perkin Trans. 2, 1995,
1415.
12. The target product may be contaminated with inorganic salts. In this case,
the solid can be purified by addition of CH2Cl2 and filtration. The filtrate is
evaporated to dryness and dried again over P2O5.
13. A high quality NaH and anhydrous conditions are crucial for the success of
this reaction. Best yields are obtained when the NaH (80% suspension in
paraffin oil, grey color) container is stored under moisture-free atmosphere.
If it is groomy and/or has pale color (or white spots), it should not be used.
14. Usually, the reaction is exothermic and the temperature rises to approximately
40◦C. If the temperature is higher, it can be kept at 40◦C with an ice-water
bath. On the other hand, if the temperature is lower and the reaction does
not start, a bath with warm water can be used to maintain the temperature at
∼40◦C.
15. If the addition is performed more rapidly and the temperature rises above
40◦C, some ethyl formate (b.p. 52–54◦C) may evaporate and the reaction
does not go to completion.
16. A byproduct with an Rf value lower than the target product may appear.
Preliminary spectroscopic analysis of this product indicates that it might be
a byproduct derived from formylation of the carbamate nitrogen.
17. If the separation between both phases is not clear, the solution should be
taken to a higher pH by addition of 2 N NaOH. The anionic form of the
product then partitions into the aqueous phase.
18. If a 60% suspension of NaH in paraffin oil is used instead of an 80% suspen-
sion, a third phase is formed that does not disappear with further additions
of NaOH (footnote #17). In this case TLC indicates that both the interphase

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and the aqueous phase contain the product, and are combined. The organic
solvent is evaporated and the aqueous residue is acidified with glacial HOAc
as described in the text. In this case, the product is not totally pure and may
be purified by silica gel flash column chromatography eluting with CH2Cl2
and increasing amounts of CH3OH (from 0 to 5%).
19. If TLC analysis shows the presence of impurities, the product may be purified
by silica gel column chromatography eluting with CHCl3 and increasing
amounts of CH3OH (0 to 5%).
20. If HCl is totally eliminated, the subsequent precipitation does not take place
properly.
Accepted April 11, 2000

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