Fmoc*: A more soluble analogue of the 9-fluorenylmethoxycarbonyl protecting group

Full text of DOI:10.1021/jo9915832

3
858
J . Org. Chem. 2000, 65, 3858-3860
F m oc*: A Mor e Solu ble An a logu e of th e
-F lu or en ylm eth oxyca r bon yl P r otectin g
Sch em e 1
9
Gr ou p
Kimberly D. Stigers, Mathew R. Koutroulis,
De Michael Chung, and J ames S. Nowick*
Department of Chemistry, University of California,
Irvine, California 92697-2025
Received October 12, 1999
The 9-fluorenylmethoxycarbonyl (Fmoc) group has
enjoyed tremendous popularity as a protecting group for
amines in the synthesis of peptides and related com-
pounds because of its stability to acid and lability to
base.1 The poor solubilities of many Fmoc-protected
amino acids and other compounds in organic solvents
offset its merits, as do difficulties in removing the
byproduct of its deprotection.2 We became painfully
cognizant of the notorious solubility problems of the Fmoc
group when we tried to use it in the synthesis of a series
of peptidomimetic compounds. To solve this problem, we
have developed the 2,7-di-tert-butyl-Fmoc group as a
more soluble analogue of the Fmoc group. We have
nicknamed this group Fmoc* to reflect its relationship
to the Fmoc group. This paper describes its preparation
and use.
roughly 25 min after the paraformaldehyde is added, but
this color change does not appear to indicate completion
of the reaction (in contrast to that reported for the
1
conversion of fluorene to 9-fluorenylmethanol). By
H
NMR spectroscopic monitoring of this reaction, we found
that the optimal time for quenching the reaction is after
5
0 min. In the third step, the hydroxymethylated com-
pound (3) is converted to Fmoc*-Cl by treatment with
1
phosgene following the procedure of Carpino and Han.
We have found the reaction of 3 to be considerably slower
than that reported by Carpino and Han for the reaction
of 9-fluorenylmethanol (3 days at 24 °C vs 4 h at 0 °C).
Fmoc*-protected compounds exhibit up to 2 orders of
5
magnitude greater solubility than their Fmoc analogues.
Like Fmoc, the Fmoc* protecting group is readily
introduced as its chloroformate. The chloroformate (Fmoc*-
Cl, 1) is prepared in three steps from fluorene, as shown
in Scheme 1. In the first step, the two tert-butyl groups
are added by Friedel-Crafts alkylation. The di-tert-
butylation of fluorene had been previously reported by
Buu-Hoi and Cagniant. Their procedure, which involved
Fmoc-protected peptidomimetic building block 4 has low
solubility in common volatile organic solvents, such as
chloroform, tetrahydrofuran, methanol, and ether, while
Fmoc*-protected analogue 5 is 1-2 orders of magnitude
more soluble. Table 1 illustrates the solubilities of these
compounds in these solvents. Fmoc*-protected p-meth-
oxybenzylamine 7 is also more soluble than its Fmoc
analogue 6; however, the improvements in solubility are
less dramatic (Table 2).
treating a neat mixture of fluorene and tert-butyl chloride
3
with AlCl
3
, proved capricious. We have found that using
carbon disulfide as solvent and the milder catalyst FeCl
3
renders the reaction reproducible and affords di-tert-
butylfluorene 2 in high yield. The hydroxymethyl group
is added in the second step. This step involves the lith-
iation of 2 and its reaction with paraformaldehyde by a
procedure analogous to Chong, Lajoie, and Tjepkema’s
4
conversion of fluorene to 9-fluorenylmethanol. This
reaction is the most difficult of the three. Only 1.0 equiv
of n-BuLi should be used; when we used an excess of 0.1
equiv, we obtained substantially diminished yields of
hydroxymethyl product 3, along with the fulvene elimi-
nation product and recovered di-tert-butylfluorene 2. The
reaction mixture sometimes undergoes a color change
The protection and deprotection of amines and related
nitrogen bases with Fmoc* is just like their protection
and deprotection with Fmoc. Amines can be protected by
(1) Carpino, L. A.; Han, G. Y. J . Org. Chem. 1972, 22, 3404-3409.
2) Kocienski, P. J . Protecting Groups; Thieme: New York, 1994; p
(
2
8
02.
(5) Previous experience has shown that di-tert-butylation can dra-
matically increase the solubilities of aromatic compounds with poor
solubilities. Nowick, J . S.; Ballester, P.; Ebmeyer, F.; Rebek, J ., J r. J .
Am. Chem. Soc. 1990, 112, 8902-8906.
(
3) Buu-Hoi, N. P.; Cagniant, P. Chem. Ber. 1944, 77, 121-126.
(
4) Chong, J . M.; Lajoie, G.; Tjepkema, M. W. Synthesis 1992, 819-
20.
1
0.1021/jo9915832 CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/24/2000

Notes
J . Org. Chem., Vol. 65, No. 12, 2000 3859
as indicator and toluene as solvent.11 p-Methoxybenzylamine
was distilled prior to use. All reactions were performed under
nitrogen; in the Friedel-Crafts and phosgenation reactions, the
nitrogen line was vented to a bubbler to prevent contaminating
the nitrogen manifold with HCl. Reactions were stirred magneti-
cally, unless otherwise indicated. Solvents were removed by
rotary evaporation under reduced pressure.
Ta ble 1. Solu bility of F m oc a n d F m oc* Der iva tives 4
a n d 5 in Com m on Or ga n ic Solven ts
solubility of 4
solubility of 5
solvent
(mg/mL)
(mg/mL)
CHCl3
THF
methanol
ether
1
8
>100
>110
0.2
<0.2
2
1
,7-Di-ter t-bu tylflu or en e (2).3 An ice-cooled, 2-L, three-
2
necked, round-bottomed flask equipped with a nitrogen inlet
adapter, a rubber septum, and a mechanical stirrer was charged
Ta ble 2. Solu bility of F m oc a n d F m oc* Der iva tives 6
a n d 7 in Com m on Or ga n ic Solven ts
with fluorene (25.00 g, 150.4 mmol), CS
2 3
(100 mL), and FeCl
(2.44 g, 15.0 mmol). 2-Chloro-2-methylpropane (34.4 mL, 316
solubility of 6
(mg/mL)
solubility of 7
(mg/mL)
mmol) was added via syringe over 5 min. After 10 min, the ice
bath was removed, and the reaction mixture was stirred for 3.5
h. Water (100 mL) was then added, and the resulting mixture
solvent
CHCl3
THF
methanol
ether
>110
>130
4.5
>270
>180
6
was partitioned between CH
100 mL). The aqueous layer was extracted with additional
CH Cl (50 mL), and the combined organic layers were washed
successively with saturated aqueous NaHCO (100 mL) and
saturated aqueous NaCl (75 mL), dried over MgSO , and
2 2
Cl (200 mL) and 1 M aqueous HCl
(
2
2
6
50
3
4
treatment with Fmoc*-Cl in a biphasic mixture of di-
chloromethane and aqueous sodium carbonate and can
concentrated to yield 40.31 g of a yellow solid. The residue was
dissolved in 200 mL of hexanes, and the solution was filtered
through a column of silica gel (9 cm h × 8 cm d) with the aid of
an additional 1 L of hexanes. The filtrate was concentrated to
yield 38.82 g (93%) of 2,7-di-tert-butylfluorene (2) as a white
solid, which was used in the next reaction without further
purification. An analytical sample was recrystallized from
6
be deprotected with a 20% solution of piperidine in DMF.7
An added benefit of Fmoc* is that the piperidine adduct
that forms upon deprotection (8) is highly lipophilic,
allowing its easy removal. This adduct can be removed
from a DMF solution of the deprotected amine by
extraction several times with hexanes.8 Alternatively,
it can be removed by dissolving the deprotected amine
in dimethyl sulfoxide, a solvent in which 8 is insoluble.
3
1
ethanol: mp 120-122 °C (lit. mp 122 °C); H NMR (500 MHz,
,9
CDCl
3
) δ 7.66 (d, J ) 8.0 Hz, 2 H), 7.56 (s, 2 H), 7.38 (d, J ) 8.1
Hz, 2 H), 3.86 (s, 2 H), 1.37 (s, 18 H); C NMR (500 MHz, CDCl )
1
3
3
δ 149.4, 143.3, 139.1, 123.8, 121.9, 119.1, 37.1, 34.8, 31.6.
4
2
,7-Di-ter t-bu tyl-9-flu or en ylm eth a n ol (3). An ice-cooled,
2
50-mL, three-necked, round-bottomed flask equipped with a
nitrogen inlet adapter, a glass stopper, a rubber septum, and a
magnetic stirring bar was charged with 2,7-di-tert-butylfluorene
(2) (10.00 g, 35.9 mmol) and THF (150 mL). A solution of
n-butyllithium in hexanes (24.4 mL, 1.47 M, 35.9 mmol) was
injected over 5 min via syringe. After an additional 3 min, finely
pulverized paraformaldehyde (1.19 g, 39.6 mmol) was added in
a single portion, the ice bath was removed, and the reaction
mixture was stirred for 50 min. The reaction was then quenched
3
by adding saturated aqueous NaHCO (120 mL) and extracted
In summary, Fmoc* alleviates solubility and byprod-
uct-removal problems associated with the Fmoc protect-
ing group. Fmoc*-Cl is easy to synthesize and can readily
be used in place of Fmoc-Cl. We anticipate that Fmoc*
will prove popular as an alternative to Fmoc.
with ether (120 mL + 3 × 50 mL). The combined organic layers
were washed with saturated aqueous NaCl (100 mL), dried over
4
MgSO , filtered, and concentrated to yield 10.87 g of white
crystals. Recrystallization from hexanes afforded 6.96 g (63%)
of 2,7-di-tert-butyl-9-fluorenylmethanol (3) as white prisms: mp
-
1 1
1
19-120 °C; IR (KBr) 3350 (br) cm ; H NMR (500 MHz, CDCl
3
)
δ 7.66 (d, J ) 8.0 Hz, 2 H), 7.61 (s, 2 H), 7.41 (dd, J ) 8.0, 1.6
Hz, 2 H), 4.07 (appar s, 3 H), 1.52 (br s, 1 H), 1.38 (s, 18 H);
1
3
Exp er im en ta l Section
C
NMR (500 MHz, CDCl ) δ 149.9, 144.3, 138.9, 124.7, 121.4, 119.3,
3
Ma ter ia ls a n d Meth od s. Commercial-grade reagents and
solvents were used without further purification except as
+
6
3
5.3, 50.5, 34.9, 31.6; HRMS (CI) m/z for C22
H
28O (M ) calcd
28O: C, 85.66;
08.2140, found 308.2144. Anal. Calcd for C22
H
indicated. CH
2
Cl
2
and THF were dried prior to use by percolation
as described by Grubbs and co-
H, 9.15. Found: C, 85.71; H, 9.22.
,7-Di-ter t-bu tyl-9-flu or en ylm eth oxyca r bon yl ch lor id e
1). An ice-cooled, 250-mL, three-necked, round-bottomed flask
through anhydrous Al
2
O
3
2
1
0
workers. Fluorene was recrystallized from ethanol. n-Butyl-
(
lithium was titrated with 2-butanol using 1,10-phenanthroline
equipped with a nitrogen inlet adapter, a glass stopper, a rubber
septum, and a magnetic stirring bar was charged with 3 (6.75
g, 21.9 mmol), CH Cl (20 mL), and a solution of phosgene in
2 2
(
6) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis; J ohn Wiley & Sons: New York, 1999; pp 506-507.
7) Periodic TLC monitoring of the deprotection of 6 and 7 revealed
toluene (33.2 mL, 1.98 M, 65.7 mmol). The ice bath was allowed
to melt, and the reaction mixture was stirred for 72 h. Concen-
tration of the reaction mixture yielded 8.13 g (100%) of Fmoc*-
Cl (1) as a light brown oil of sufficient purity for use. An
analytical sample was obtained as a white solid by adding a
minimal amount of pentane, chilling to -78 °C under nitrogen
until crystals formed, decanting the mother liquor, and removing
residual pentane under vacuum: mp 63-65 °C; IR (KBr) 1780
(
that the cleavage of the Fmoc* group proceeds at almost the same rate
as the Fmoc group. Complete conversion of the Fmoc* derivative 7 to
p-methoxybenzylamine with 20% v/v piperidine in DMF requires 20
min while the Fmoc derivative 6 requires 15 min. The slightly slower
rate of the Fmoc* compound may arise from electron donation by the
tert-butyl substituents on fluorene.
(
8) UV analysis of the DMF and hexane layers demonstrated that
approximately 75% of the piperidine adduct 8 is removed with each
extraction by an equal volume of hexane. After four hexane washes,
1% of 8 remains in the DMF layer.
(
efficient when the deprotected amine contains highly polar functional
groups, such as amides. Lipophilic amines can partition into the
hexanes phase resulting in a decreased yield. Deprotection of 7 followed
by washing of the DMF layer with four portions of hexanes gave only
a 60% yield of p-methoxybenzylamine. Some of the p-methoxybenzyl-
amine was lost by partitioning into the hexanes layer.
-
1
1
cm ; H NMR (400 MHz, CDCl
7
3
) δ 7.65 (d, J ) 8.0 Hz, 2 H),
.60 (s, 2 H), 7.44 (dd, J ) 8.2, 2.0 Hz, 2 H), 4.54 (d, J ) 8.0 Hz,
<
1
3
9) The removal of the Fmoc* deprotection byproduct (8) is most
2 H), 4.26 (t, J ) 7.6 Hz, 1 H), 1.37 (s, 18 H); C NMR (400
MHz, CDCl ) δ 150.7, 150.2, 142.4, 138.7, 125.3, 122.0, 119.5,
3
+
7
3
3.9, 46.1, 34.9, 31.5; HRMS (CI) m/z for C23
H
27
O
2
Cl (M ) calcd
70.1699, found 370.1700. Anal. Calcd for C23
H
27
2
O Cl: C, 74.48;
H, 7.34. Found: C, 74.87; H, 7.41.
(
10) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. A.; Rosen, R. K.;
(11) Watson, S. C.; Eastham, J . F. J . Organomet. Chem. 1967, 9,
165-168.
Timmers, F. J . Organometallics 1996, 15, 1518-1520.

3
860 J . Org. Chem., Vol. 65, No. 12, 2000
Notes
Rep r esen ta tive P r oced u r e for th e F m oc*-P r otection of
mg, 0.270 mmol). A solution of 20% (v/v) piperidine in DMF (2.7
mL) was added in one portion, and the mixture was stirred at
room temperature for 20 min. The solution was diluted with 5
mL of DMF and extracted 4 × 10 mL with hexanes. The DMF
a n Am in e. An ice-cooled, two-necked, round-bottomed flask
equipped with a magnetic stirring bar, nitrogen inlet adapter,
and septum was charged with p-methoxybenzylamine (0.422 g,
3
(
.07 mmol), dichloromethane (1.0 mL), and 10% aqueous Na
8.3 mL). After 5 min, a solution of Fmoc*-Cl (1.14 g, 3.07 mmol)
in CH Cl (3.2 mL) was added over 2 min. The ice bath was
2
CO
3
layer was concentrated to yield 53.4 mg (94%) of 2-MeO-5-NO -
2
C H -CONHNH as a white solid.
6
3
2
2
2
removed, and the reaction mixture was stirred at room temper-
ature for 2 h. The reaction mixture was diluted with 60 mL of
6
Ack n ow led gm en t. The authors thank the NIH and
CH
was extracted with CH
organic layers were dried over MgSO
to yield 1.38 g of white foam. Purification by column chroma-
tography on silica gel (1:3 EtOAc/hexanes) yielded 1.28 g (88%)
of 7 as a white solid.
Rep r esen ta tive P r oced u r e for th e F m oc*-Dep r otection
a n d Rem ova l of Byp r od u ct 8 by Extr a ction . A pear-shaped
flask equipped with a magnetic stirring bar and nitrogen inlet
2
Cl
2
and washed with 60 mL of 1 M HCl. The aqueous layer
Cl
(2 × 20 mL), and the combined
, filtered, and concentrated
NSF for grant support (GM-49076 and CHE-9813105).
J .S.N. thanks the following agencies for support in the
form of awards: the Camille and Henry Dreyfus Foun-
dation (Teacher-Scholar Award), the Alfred P. Sloan
Foundation (Alfred P. Sloan Research Fellowship), and
the American Chemical Society (Arthur C. Cope Scholar
Award).
2
2
4
was charged with 2-MeO-5-NO
2
-C
6
H
3
-CONHNH-Fmoc* (5, 147
J O9915832