Solid-phase synthesis of peptidyl thioacids employing a 9-fluorenylmethyl thioester-based linker in conjunction with Boc chemistry

Article abstract of DOI:10.1021/jo901218g

(Chemical Equation Presented) A method for the synthesis of peptidyl thioacids is described on the basis of the use of the N-[9-(thiomethyl)-9H- fluoren-2-yl]succinamic acid and cross-linked aminomethyl polystyrene resin. The method employs standard Boc chemistry SPPS techniques in conjunction with 9-fluorenylmethyloxycarbonyl protection of side-chain alcohols and amines and 9-fluorenylmethyl protection of side-chain acids and thiols. Cleavage from the resin is accomplished with piperidine, which also serves to remove the side-chain protection and avoids the HF conditions usually associated with the resin cleavage stage of Boc chemistry SPPS. The so-obtained thioacids are converted to simple thioesters in high yield by standard alkylation according to well-established methods. 2009 American Chemical Society.

Full text of DOI:10.1021/jo901218g

pubs.acs.org/joc
Solid-Phase Synthesis of Peptidyl Thioacids Employing a
9-Fluorenylmethyl Thioester-Based Linker in Conjunction with
Boc Chemistry
David Crich†,‡,* and Kasinath Sana‡
†Centre de Recherche de Gif-sur-Yvette, Institut de Chimie des Substances Naturelles, Centre National de la
Recherche Scientifique, avenue de la Terrasse, 91198 Gif-sur-Yvette, France, and ‡Department of Chemistry,
Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202
dcrich@icsn.cnrs-gif.fr
Received June 9, 2009
A method for the synthesis of peptidyl thioacids is described on the basis of the use of the
N-[9-(thiomethyl)-9H-fluoren-2-yl]succinamic acid and cross-linked aminomethyl polystyrene resin.
The method employs standard Boc chemistry SPPS techniques in conjunction with 9-fluorenyl-
methyloxycarbonyl protection of side-chain alcohols and amines and 9-fluorenylmethyl protection
of side-chain acids and thiols. Cleavage from the resin is accomplished with piperidine, which also
serves to remove the side-chain protection and avoids the HF conditions usually associated with the
resin cleavage stage of Boc chemistry SPPS. The so-obtained thioacids are converted to simple
thioesters in high yield by standard alkylation according to well-established methods.
Introduction
employed in amide bond forming reactions either directly3 or
indirectly through their facile conversion to thioesters, key
intermediates in a variety of chemical and enzymic amide
ligation processes.4
The instability of thioesters toward the typical conditions
of 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry solid-
phase peptide synthesis (SPPS), particularly the treatment
Thioacids [RC(dO)SH]1 are a fascinating but underap-
preciated class of compounds with a unique reactivity pro-
file2 that arises in part from their pKa and the consequent
ability of the conjugate base to act in a highly selective
manner as nucleophile in aqueous media at pH 3-6. Not
surprisingly, therefore, most applications of thioacids have
been in the field of peptide chemistry where they have been
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2000, 69, 923–960. (c) Yeo, D. S. Y.; Srinivasan, R.; Chen, G. Y. J.; Yao, S. Q.
Chem.;Eur. J. 2004, 10, 4664–4672. (d) Macmillan, D. Angew. Chem., Int.
Ed. 2006, 45, 7668–7672. (e) Hackenberger, C. P. R.; Schwarzer, D. Angew.
Chem., Int. Ed. 2008, 47, 10030–10074. (f) Kent, S. B. H. Chem. Soc. Rev.
2009, 38, 338–351. (g) Flavell, R. R.; Muir, T. W. Acc. Chem. Res. 2009, 42,
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DOI: 10.1021/jo901218g
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Published on Web 09/02/2009
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2009 American Chemical Society

Crich and Sana
JOCArticle
with organic bases employed in the cleavage of Fmoc groups,
led to an initial reliance on tert-butyloxycarbonyl-based (Boc)
chemistry for the synthesis of peptidyl thioesters,4o,q,5 but the
HF conditions typically required for cleavage from the resin
following Boc chemistry SPPS limit the use of this chemis-
try. Fmoc chemistry-based methods have subsequently been
developed that replace piperidine in the Fmoc removal step
by cocktails of 1-methylpyrrolidine, hexamethylenimine, and
1-hydroxybenzotriazole, but it has been found that these
methods are prone to racemization at the thioester position.6
The backbone amide linker (BAL) strategy enables Fmoc
chemistry SPPS with subsequent incorporation of a C-terminal
thioester prior to cleavage from the resin but requires careful
control of reaction conditions to avoid epimerization on
introduction of the thioester to the C-terminal end of the
peptide chain.7 To circumvent these problems, numerous
methods have been developed according to which, after com-
pletion of the peptide synthesis by Boc or Fmoc methods, the
linker to the resin is activated in such a way as to permit its
displacement by a thiol or thiolate resulting in the liberation of
the peptide in the form of the desired thioester or thioacid.8
A variant on the BAL strategy, which avoids the epimerization
problem, carries a C-terminal trithioorthoester through the
Fmoc chemistry SPPS sequence before converting it to the
required thioester by controlled hydrolysis.9 More recently, a
number of strategies have been developed in which thioesters
are generated by O-S or N-S shifts of mercapto esters and
mercapto amides following unmasking of a protected thiol
group.10 Despite the considerable ingenuity that has been
deployed in the development of the above methods, none
combine the directness that obviously results from the use of
a simple C-terminal thioester-based linker with a method for
release from the resin that avoids the use of HF. Previously, we
introduced the 9-fluorenylmethyl thioesters from which thio-
acids are liberated by simple treatment with piperidine, that is,
under the conditions usually employed for the cleavage of
Fmoc groups in Fmoc chemistry SPPS.11 We conceived that a
linker based on the 9-fluorenylmethyl thioester would be compa-
tible with the general conditions of Boc chemistry SPPS and that
following peptide assembly treatment of the resin with piperidine
would release the Boc-protected peptide into solution in the form
of a C-terminal thioacid that could be readily transformed into a
thioester by simple alkylation. Of essence, this method, whose
reduction to practice we report here, employs conditions no more
forcing than those encountered in standard Boc and Fmoc
chemistry SPPS protocols and circumvents the terminal HF
treatment that limits most Boc chemistry SPPS methods. We
further conceived that the utility of this method would be
enhanced by the application of side-chain protection strategies
involving either a third orthogonal system enabling retention of
side-chain protecting groups post cleavage12 or a system accord-
ing to which all protecting groups would be removed concomi-
tantly with cleavage of the thioacid from the resin, depending on
the ultimate application envisaged for the peptidyl thioacid.
Results and Discussion
The mercapto functionalized linker, N-[9-(tritylthiomethyl)-
9H-fluoren-2-yl]succinamic acid (5), was prepared from com-
mercially available 9H-fluoren-2-amine as shown in Scheme 1.
The synthesis began with the conversion of 9H-fluoren-2-amine
to the corresponding hydroxyl functional compound 1 follow-
ing a literature procedure13 involving formylation followed by
reduction. Tosylation of 1 under standard conditions gave the
sulfonate 2, from which the amine 3 was liberated with
trifluoroacetic acid. Reaction of 3 with succinic anhydride
provided the hemisuccinate 4 that was subjected to treatment
with tritylmercaptan and Hunig’s base to give the protected
linker 5 in excellent yield (Scheme 1). Treatment of 5 with
diisopropyl carbodiimide and N-hydroxybenzotriazole in DMF
gave an activated intermediate that was allowed to react with
1% divinylbenzene cross-linked aminomethylpoystyrene resin
(0.41 mmol/g loading). Following washing with DMF, the
functionalized resin was exposed to a 50% solution of TFA in
dichloromethane to yield the desired resin-bound 9-fluorenyl-
methylthiol derivative 6 (Scheme 1). The attachment of linker 5
to the aminomethylpolystyrene resin was also accomplished in
a satisfactory manner with the O-benzotriazolyltetramethyl-
uronium hexafluorophosphate (HBTU) reagent14 with the aid
of diisopropylethylamine as base.
(5) (a) Canne, L. E.; Walker, S. M.; Kent, S. B. H. Tetrahedron Lett. 1995,
36, 1217–1220. (b) Canne, L. E.; Ferre- D’Amare, A. R.; Burley, S. K.; Kent,
S. B. H. J. Am. Chem. Soc. 1995, 117, 2998–3007. (c) Lu, W.; Qasim, M. A.;
Kent, S. B. H. J. Am. Chem. Soc. 1996, 118, 8518–8523. (d) Hojo, H.;
Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111–117. (e) Kawakami, T.;
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Houghten, R. A. J. Comb. Chem. 2008, 10, 613–616.
The preparation of a series of suitably protected amino acids
was then undertaken. Thus, N-tert-butoxycarbonyl-L-serine and
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8669–8672. (b) Hasegawa, K.; Sha, Y. L.; Bang, J. K.; Kawakami, T.; Akaji,
K.; Aimoto, S. Lett. Pept. Sci 2002, 8, 277–284.
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Barany, G. J. Am. Chem. Soc. 1998, 120, 5441–5452. (b) Alsina, J.; Yokum,
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J.; Yokum, T. S.; Albericio, F.; Barany, G. Tetrahedron Lett. 2000, 41, 7277–
7280.
(11) Crich, D.; Sana, K.; Guo, S. Org. Lett. 2007, 9, 4423–4426.
(12) The use of allyl esters in alloxycarbamates as a protecting group
system orthogonal with both Boc and Fmoc chemistries is widely established.
(a) Grieco, P.; Gitu, P. M.; Hruby, V. J. J. Peptide. Res. 2001, 57, 250–256.
(b) Kates, S. A.; Daniels, S. B.; Albericio, F. Anal. Biochem. 1993, 212, 303–
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1269–1270. (b) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem.
Soc. 1999, 121, 11369–11374. (c) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent,
S. B. H.; Ellman, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121, 11684–
11689. (d) Sweing, A.; Hilvert, D. Angew. Chem., Int. Ed. 2001, 40, 3395–
3396. (e) Camarero, J. A.; Hackel, B. J.; De Yoreo, J. J.; Mitchell, A. R.
J. Org. Chem. 2004, 69, 4145–4151. (f) Blanco-Canosa, J. B.; Dawson, P. E.
Angew. Chem., Int. Ed. 2008, 47, 6851–6855. (g) Yamamoto, N.; Tanabe, Y.;
Okamoto, R.; Dawson, P. E.; Kajihara, Y. J. Am. Chem. Soc. 2008, 130, 501–
510.
310. (c) Kates, S. A.; Sole, N. A.; Johnson, C. R.; Hudson, D.; Barany, G.;
Albericio, F. Tetrahedron Lett. 1993, 34, 1549–1552. (d) Bloomberg, G. B.;
Askin, D.; Gargaro, A. R.; Tanner, M. A. J. Tetrahedron Lett. 1993, 34,
4709–4712. Alternative possibilities include the nitrobenzenesulfonyl pro-
tecting group for amines. (e) Kan, T.; Fukuyama, T. Chem. Commun. 2004,
353–359. (f) Halpin, D. R.; Lee, J. A.; Warren, S. J.; Harbury, P. B. PLOS
Biol. 2004, 2, 1031–1038.
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Grandas, A.; Marchan, V.; Pastor, J. J.; Pedroso, E.; Rabanal, F.; Royo,
M. Synth. Commun. 2001, 31, 225–232.
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(10) (a) Botti, P.; Villain, M.; Manganiello, S.; Gaertner, H. Org. Lett. 2004,
6, 4861–4864. (b) Warren, J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J.
J. Am. Chem. Soc. 2004, 126, 6576–6578. (c) George, E. A.; Novick, R. P.; Muir,
T. W. J. Am. Chem. Soc. 2008, 130, 4914–4924. (d) Kawakami, T.; Aimoto, S.
Tetrahedron 2009, 65, 3871–3877.
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19, 1269–1272. (b) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D.
Tetrahedron Lett. 1989, 30, 1927–1930. (c) Fields, G. B.; Tian, Z.; Barany, G. In
Synthetic Peptides: A User’s Guide; Grant, G. A., Ed.; Freeman: New York,
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SCHEME 1. Preparation of a Functionalized Resin
SCHEME 2. Preparation of Protected Hydroxy Amino Acids
the analogous L-threonine and L-tyrosine derivatives were
converted to their allyl esters with potassium carbonate and
then to the 9-fluorenylmethyl carbonates with Fmoc chloride
and pyridine. A combination of palladium(II) acetate, triphe-
nylphosphine, and phenylsilane was the reagent of choice for
the liberation the allyl esters required to complete this short
sequence (Scheme 2).
THF at reflux for 24 h, then with 9-fluorenylmethanol,
dicyclohexylcarbodiimide, and 4-dimethylaminopyridine,
and finally with gaseous hydrogen chloride gave the
mono esters 13 and 14. These HCl salts were then con-
verted to the N-Boc derivatives in the standard manner
(Scheme 3).
The corresponding mono allyl esters 17 and 18 were
accessed according to the literature method,16 as were the
9-fluorenylmethyl thioether of Boc-L-cysteine 19,15 the
Following a literature protocol,15 treatment of powder-
ed L-aspartic and L-glutamic acids with triethylborane in
(15) Albericio, F.; Nicolas, E.; Rizo, J.; Ruiz-Gayo, M.; Pedroso, E.;
Giralt, E. Synthesis 1990, 119–122.
(16) Webster, K. L.; Maude, A. B.; O’Donnell, M. E.; Mehrotra, A. P.;
Gani, D. J. Chem. Soc., Perkin Trans. 1 2001, 1673–1695.
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SCHEME 3. Preparation of Mono-9-fluorenylmethyl Esters of Aspartic and Glutamic Acid
Fmoc-protected L-arginine derivative 20,17 and Alloc-pro-
tected L-histidine 21.18
Human Secretory Phospholipase A2 (hsPLAA2),21 and peptide
27 (Table 1, entry 5) is the 65-84 unit of Human Parathyroid
Hormone (hPTH).22
1
The H and 13C NMR spectra of the crude tetrapeptidyl
thioacid 22 as obtained on simple release from the resin,
acidification with HCl, and drying are presented in the
Supporting Information (Figures 1 and 2) to illustrate the
high degree of purity typically obtained by this method. In a
similar vein, the ESI-TOF mass spectrum of the peptidyl
thioacid 27, immediately after release from the resin and
prior to purification by HPLC is presented in the Supporting
Information as Figure 3.
Particular attention is drawn to entries 4 and 5 of Table 1
in which the C-terminal amino acid is asparagine and
glutamine, respectively. The amino acid building blocks for
these residues were employed without protection of the side-
chain amide functionality, and it is noteworthy that cycliza-
tion of these amides onto the resin-bound thioester with
premature peptide release in the form of an imide did
not occur to any significant extent.23 In general, the strategy
of employing Fmoc protection for side-chain amines and
hydroxyl groups, coupled with the protection of side-chain
carboxylates and thiols, ensures clean chemistry while elim-
inating the need for extra deprotection steps pre- or post-
cleavage of the peptidyl thioacid from the resin. Neverthe-
less, should the retention of side-chain protection be required
on cleavage from the resin, this may be conveniently accom-
plished through the use of building blocks whose side chains
are covered with either the allyl or allyloxycarbonyl system12
depending on the case (Table 1, entries 2 and 3).
By way of example, two of the peptidyl thioacids obtained
in this manner were converted to the corresponding S-benzyl
thioesters by simple alkylation with benzyl bromide and sym-
collidine in DMF (Scheme 4).5c
Finally, as a demonstration of the broad scope of the
chemistry of thioacids, a single decapeptidyl thioacid was
subjected to reaction with a sulfonyl azide, under conditions
described by the Williams and Liskamp groups24 for much
With all building blocks in hand, attention was turned to
SPPS using standard Boc techniques with DIC/HOBt as the
coupling agent and TFA to liberate the N-terminus of the
growing chains from their Boc derivatives. A number of pep-
tides were assembled in this manner as shown in Table 1
(entries 1-5). As with the preparation of the resin-bound thiol
6 (Scheme 1), this methodology is not limited to carbodiimide
chemistry but is perfectly adaptable to the other methods as
evidenced by the application of the HBTU protocol (Table 1,
entry 6). After completion of the on-resin procedure, treatment
with a solution of piperidine in DMF or, to enable direct loading
of the reaction mixture to the HPLC column, acetonitrile
released the desired thioacids protected at the N-terminal ends
in the form of the Boc derivatives,19 which were typically
obtained with a high degree of purity as determined by ESI-
TOF and HPLC methods. Similar treatment of individual beads
was used to systematically monitor the individual reaction steps
during the course of the peptide assembly sequence. Although
mass spectrometry was the method of choice for monitoring
these SPPS reactions, the Kaiser ninhydrin test also performed
in a perfectly satisfactory manner for both the coupling and Boc
removal steps. While the thioacid 22 was a simple model
tetrapeptidyl thioacid (Table 1, entry 1), the peptidyl thioacids
23, 24, 25, and 27 are all natural sequences. The sequences of
thioacids 23 and 24 (Table 1, entries 2 and 3) were selected from
the Glucagon-like peptide-1 (GLP-1)20 and represent segments
7-16 and 17-26, respectively, of that peptidyl hormone. Pep-
tide 25 (Table 1, entry 4) represents the 94-101 segment of
(21) Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 10068–10073.
(22) Fairwell, T.; Hospattankar, A. V.; Ronan, R.; Brewer, H. B., Jr;
Chang, J. K.; Shimizu, M.; Zitzner, L.; Arnaud, C. D. Biochemistry 1983, 22,
2691–2697.
(23) This is evident simply from the yields of the isolated thioacids 25 and
27, which require average minimum coupling yields of >93% and >97%,
respectively, for each coupling deprotection cycle. For comparable reasons
premature peptide cleavage by diketopiperazine formation at the level of
deprotection of the dipeptide and the migration of side-chain protecting
groups to N-terminal amines on neutralization do not appear to be major
concerns, at least for the examples provided.
(24) (a) Shangguan, N.; Katukojvala, S.; Greenberg, R.; Williams, L. J.
J. Am. Chem. Soc. 2003, 125, 7754–7755. (b) Merkx, R.; Brouwer, A. R.;
Rijkers, D. T. S.; Liskamp, R. M. J. Org. Lett. 2005, 7, 1125–1128. (c) Barlett,
K. N.; Kolakowski, R. V.; Katukojvala, S.; Williams, L. J. Org. Lett. 2006, 8,
823–826. (d) Kolakowski, R. V.; Shangguan, N.; Sauers, R. R.; Williams,
L. J. J. Am. Chem. Soc. 2006, 128, 5695–5702. (e) Merkx, R.; Van Haren,
M. J.; Rijkers, D. T. S.; Liskamp, R. M. J. J. Org. Chem. 2007, 72, 4574–4577.
(17) Katzhendler, J.; Klauzner, Y.; Beylis, I.; Mizhiritskii, M.; Shpernat,
Y.; Ashkenazi, B.; Fridland, D. PCT Int. Appl. 076744, 2005.
(18) Dangles, O.; Guibe, F.; Balavoine, G.; Lavielle, S.; Marquet, A.
J. Org. Chem. 1987, 52, 4984–4993.
(19) The peptides were isolated in the form of their N-Boc derivatives
simply owing to the use of Boc-protected building blocks. Use of Fmoc-
protected builing blocks in the final coupling step will necessarily yield
peptides with the N-terminus unprotected.
(20) Lee, S.-H.; Lee, S.; Youn, Y. S.; Na, D. H.; Chae, S. Y.; Byun, Y.;
Lee, K. C. Bioconjugate Chem. 2005, 16, 377–382.
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TABLE 1. Boc Chemistry SPPS of Peptidyl Thioacidsa
aAll reactions used ∼0.1 mmol of aminomethyl polystyrene resin (244 mg, resin loading 0.41 mmol/g). All cleavage steps used 25% TFA in CH2Cl2
(2 ꢀ 1.5 mL, 2 ꢀ 30 min) after which the resin was washed with CH2Cl2 (3 ꢀ 2 mL) and neutralized with 5% DIPEA in CH2Cl2 (5 mL). All coupling
reactions with the exception of entry 6i used Boc-L-amino acids (0.4 mmol) preactivated with HOBt (54 mg, 0.4 mmol) and DIC (62 μL, 0.4 mmol) in
DMF (1 mL) for 30 min. The preactivated amino acid was added to the resin with an additional DMF (1 mL) and shaken for 3 h. After coupling, the resin
was washed with DMF (3 ꢀ 2 mL) and CH2Cl2 (3 ꢀ 2 mL). bWith the exception of 22, which required no purification, yields are for compounds isolated
and purified by RP-HPLC and are based on the substitution level of the aminomethyl polystyrene resin, taking into account the aliquots removed for
monitoring of reaction progress. cThe peptidyl thioacid was released from the resin by treatment with 20% piperidine in DMF for 20 min. dThe Alloc
group was cleaved from the side chain of the histidine residue in the course of the treatment with piperidine. eAfter incorporation of cysteine into the
peptide sequence, triethylsilane (0.5%) was included in the TFA/CH2Cl2 solution used for Boc-removal in all subsequent coupling steps. fThe peptidyl
thioacid was released from the resin by the treatment with 50% piperidine in acetonitrile for 30 min. Directly analogous results could be obtained with
piperidine in DMF, but the use of acetonitrile enables the reaction mixture to be loaded directly onto the HPLC for purification. gIn addition to the
desired peptide R-thioacid 25, 15% of a disulfide 26 corresponding to oxidative dimerization of thioacid was isolated by RP-HPLC. This structure is
written as a symmetric cystine derivative rather than as the alternative diacyl sulfide as no such dimers were seen in any of the other examples. hAfter
introduction of the eighth amino acid in the sequence and the removal of the Boc group it was found necessary to conduct the neutralization with a 5%
solution of DIPEA in N-methylpyrrolidine. All subsequent coupling and deprotection steps in this sequence also employed N-methylpyrrolidine as
solvent.25 iThis reaction sequence employed HBTU and iPr2NEt for the coupling reactions in place of DIC/HOBt.
SCHEME 4. Peptidyl Thioester Synthesis
SCHEME 5. Formation of a Sulfonylamide
Overall, we describe the successful implementation of a
straightforward method for the SPPS of peptidyl thioacids
using standard Boc chemistry with release from the resin
using conditions typically used for Fmoc removal during the
course of Fmoc chemistry SPPS. In conjunction with the
protection of side chain amino and hydroxyl groups as Fmoc
carbonates, and of side-chain acids and thiols as 9-fluorenyl-
simpler substrates, resulting in the isolation of a C-terminal
sulfonylamide (Scheme 5).
(25) Fields, G. B.; Fields, C. G. J. Am. Chem. Soc. 1991, 113, 4202–4207.
J. Org. Chem. Vol. 74, No. 19, 2009 7387

Crich and Sana
JOCArticle
methyl esters and thioethers this chemistry provides a very
convenient and mild means of access to peptidyl thioacids
and, by simple alkylation, of their S-esters.
1H), 7.44-7.42 (d, J = 7.2 Hz, 1H), 7.34-7.29 (m, 3H),
7.17-7.13 (t, J = 6.4 Hz, 1H), 6.86 (s, 1H), 6.71-6.68 (dd,
J = 1.6, 8.4 Hz, 1H), 4.26-4.18 (m, 2H), 4.13-4.09 (t, J = 7.2
Hz, 1H), 3.67 (br s, 2H), 2.41 (s, 3H); 13C NMR (100 MHz)
δ 146.4, 145.1, 144.7, 142.0, 141.7, 133.1, 132.3, 130.1, 128.2,
128.1, 125.7, 125.1, 121.1, 119.0, 115.2, 112.2, 72.5, 46.8, 21.9;
ESI-HRMS calcd for C21H19NO3S [M þ H]þ 366.1164, found
366.1152.
N-[9-(Tosyloxymethyl)-9H-fluoren-2-yl]succinamic Acid (4).
To a stirred solution of 3 (1.8 g, 4.9 mmol) in THF (10 mL)
was added succinic anhydride (590 mg, 5.9 mmol) portionwise
over a period of 10 min at room temperature. The reaction
mixture was stirred at room temperature for 1 h, then the
reaction mixture was concentrated and subjected to chromato-
graphic purification using 5% methanol in dichloromethane
when it afforded 4 (2.1 g, 91%): white solid, crystallized from
chloroform/hexane; mp 165.8-166.2 °C; 1H NMR (400 MHz)
δ 7.65-7.63 (m, 3H), 7.55-7.51 (m, 3H), 7.37-7.7.36 (d, J =
7.2 Hz, 1H), 7.26-7.2 (m, 4H), 7.14-7.11 (t, J = 7.2 Hz, 1H),
4.20-4.13 (m, 2H), 4.07-4.04 (t, J = 7.2 Hz, 1H), 2.67-2.62
(m, 4H), 2.32 (s, 3H); 13C NMR (100 MHz) δ 175.6, 171.1, 145.3,
143.3, 142.4, 141.1, 137.7, 137.3, 132.5, 130.1, 128.2, 128.0,
126.8, 125.1, 120.5, 120.1, 120.0, 119.8, 116.8, 72.0, 46.8, 31.7,
29.4, 21.7; ESI-HRMS calcd for C25H23NO6S [M þ Na]þ
488.1144, found 488.1120.
Experimental Section
General Methods. Unless otherwise stated, 1H and 13C NMR
were recorded in CDCl3 solution and optical rotations in CHCl3
solutions. All organic extracts were dried over sodium sulfate
and concentrated under aspirator vacuum. Chromatographic
purifications were carried out over silica gel. All peptide thioacid
syntheses were carried out on a 0.1 mmol scale employing 1%
DVB cross-linked aminomethyl polystyrene resin (244 mg, resin
loading 0.41 mmol/g) in a 10 mL manual synthesizer glass
reaction vessel with a Teflon-lined screw cap. The peptide resin
was shaken during both NR-tert-butoxycarbonyl deprotection
and coupling steps. After each coupling step, formation of the
desired peptide thioacid was confirmed by cleavage of a small
amount (∼5 mg) of resin using a 20% solution of piperidine in
DMF for 20 min, followed by examination by ESI-TOF mass
spectrometry. Isolated yields were determined on the basis of the
theoretical yield calculated for the use of 0.1 mmol of resin with a
loading of 0.41 mmol/g. These yields take no account of the aliquots
removed for monitoring and are therefore minimum yields.
[2-(tert-Butoxycarbonylamino)-9H-fluoren-9-yl]methyl 4-Methyl-
benzenesulfonate (2). To a stirred solution of [2-(tert-butoxy-
carbonylamino)-9H-fluoren-9-yl]methanol13 (1.8 g, 5.8 mmol)
and 4-methylbenzenesulfonyl chloride (1.65 g, 8.7 mmol) in
CHCl3 (20 mL) was added pyridine (0.9 mL, 11.6 mmol) at
0 °C. The reaction mixture was stirred at room temperature for
6 h, and then the organic layer was washed with 1 M HCl, water,
and brine, dried, and concentrated. Chromatographic purifica-
tion using 30% ethyl acetate in hexane afforded 2 (2.42 g, 90%):
yellowish syrup; 1H NMR (500 MHz) δ 7.78-7.76 (d, J =
8.0 Hz, 2H), 7.66-7.61 (dd, J = 8.5, 12.8 Hz, 2H), 7.57 (s, 1H),
7.51-7.50 (d, J = 7.5 Hz, 1H), 7.38-7.35 (t, J = 7.0 Hz, 2H),
7.31-7.29 (d, J = 8 Hz, 2H), 7.25-7.22 (t, J = 7.5 Hz, 1H), 6.61
(s, 1H), 4.31-4.24 (m, 2H), 4.20-4.17 (t, J = 7.5 Hz, 1H), 2.43
(s, 3H), 1.56 (s, 9H); 13C NMR (125 MHz) δ 153.0, 145.1, 143.7,
142.5, 141.3, 138.0, 136.5, 133.0, 130.1, 128.3, 128.2, 126.7,
125.3, 120.7, 119.8, 118.8, 115.6, 80.9, 72.0, 46.9, 28.6, 21.8;
ESI-HRMS calcd for C26H27NO5S [M þ Na]þ 488.1508, found
488.1486.
N-[9-(Tritylthiomethyl)-9H-fluoren-2-yl]succinamic Acid (5).
To a stirred solution of 4 (2.0 g, 4.3 mmol) and triphenylmethan-
ethiol (1.5 g, 5.4 mmol) in DMF (15 mL) was added diisopro-
pylethylamine (1.8 mL, 10.8 mmol) at 0 °C. The reaction
mixture was stirred at room temperature for 15 h, after which
time the DMF was removed under high vacuum and the crude
mixture was dissolved in EtOAc, washed with water and brine,
dried, and concentrated. Chromatographic purification of the
residue using 4% methanol in dichloromethane afforded 5
(2.23 g, 91%): light brown solid, crystallized from chloroform/
hexane; mp 84-85 °C; 1H NMR (500 MHz) δ 7.80 (br s, 1H),
7.57-7.53 (m, 4H), 7.43-7.41 (m, 6H), 7.31-7.24 (m, 8H),
7.21-7.7.17 (m, 4H), 3.57-3.54 (t, J = 7.0 Hz, 1H), 2.74-2.70
(m, 3H), 2.67-2.62 (m, 3H); 13C NMR (125 MHz) δ 177.5,
170.5, 147.2, 146.1, 144.9, 140.5, 137.5, 136.8, 130.0, 128.2,
127.7, 127.0, 126.7, 124.9, 120.4, 119.9, 119.7, 116.9, 67.6,
47.2, 36.1, 32.0, 29.7; ESI-HRMS calcd for C37H31NO3S [M þ
Na]þ 592.1922, found 592.1892.
(2-Amino-9H-fluoren-9-yl)methyl 4-Methylbenzenesulfonate
(3). To a stirred solution of 2 (2.4 g, 5.2 mmol) in CH2Cl2
(16 mL) was added TFA (4 mL) dropwise at 0 °C. The reaction
mixture was stirred at the same temperature for 20 min before it
was neutralized at 0 °C by saturated aqueous NaHCO3. The
organic layer was washed with water and brine, dried, and
concentrated. Chromatographic purification using 40% ethyl
acetate in hexane afforded 3 (1.9 g, 100%): light yellow syrup;
1H NMR (400 MHz) δ 7.78-7.76 (d, J = 8.4 Hz, 2H),
7.57-7.55 (d, J = 7.2 Hz, 1H), 7.50-7.48 (d, J = 8.0 Hz,
Acknowledgment. We thank the NIH (GM62160) for
support of this work.
Supporting Information Available: Complete experimental
1
details, copies of the H and 13C NMR spectra of compounds
2-5, 7-12, and 22-30, and HPLC traces and mass spectra of
peptidyl thioacids 22-27. This material is available free of
charge via the Internet at http://pubs.acs.org.
7388 J. Org. Chem. Vol. 74, No. 19, 2009