An α-formylglycine building block for Fmoc-based solid-phase peptide synthesis

Article abstract of DOI:10.1021/ol052623t

(Chemical Equation Presented) Nearly all known sulfatases share a common active site modification that is required for their activity: conversion of cysteine to α-formylglycine. We report the synthesis of an α-formylglycine building block suitable for Fmo

Full text of DOI:10.1021/ol052623t

ORGANIC
LETTERS
2006
Vol. 8, No. 1
131-134
An
r-Formylglycine Building Block for
Fmoc-Based Solid-Phase Peptide
Synthesis
Jason Rush‡ and Carolyn R. Bertozzi*,†,‡,§
Department of Chemistry, Department of Molecular and Cell Biology, and Howard
Hughes Medical Institute, UniVersity of California, Berkeley, California 94720
crb@berkeley.edu
Received October 28, 2005
ABSTRACT
Nearly all known sulfatases share a common active site modification that is required for their activity: conversion of cysteine to
We report the synthesis of an -formylglycine building block suitable for Fmoc-based solid-phase peptide synthesis. The building block was
incorporated into a synthetic peptide derived from the active site of a Mycobacterium tuberculosis sulfatase.
r-formylglycine.
r
Sulfation of biomolecules is a common modification that is
involved in both normal and pathological processes.1 For
example, sulfated oligosaccharides play important roles in
inflammation,2 tyrosine sulfation is linked to the recognition
of target cells by HIV-1,3 and an abundance of sulfated
glycolipids is correlated with Mycobacterium tuberculosis
strain virulence.4
geminal diol (2) acts as a nucleophile to produce a sulfated
enzyme intermediate (3) (Figure 1). This intermediate then
collapses, expelling inorganic sulfate and regenerating FGly.
Two classes of enzymes control sulfation. Sulfotransferases
catalyze the formation of sulfate esters,5 while sulfatases
catalyze their hydrolysis.6 Sulfatases utilize a unique mech-
anism that is facilitated by an unusual residue in the active
site, R-formylglycine (FGly) (1) (Figure 1). In the proposed
mechanism, FGly is hydrated in the active site7 and the
* Address correspondence to this author.
† Department of Molecular and Cell Biology.
‡ Department of Chemistry.
Figure 1. Catalytic cycle of sulfatases.
§ Howard Hughes Medical Institute.
(1) Hemmerich, S.; Verdugo, D.; Rath, V. L. Drug DiscoV. Today 2004,
9, 967-975.
FGly is installed in the sulfatase active site via a post-
translational modification prior to folding.8 A genetically
encoded cysteine residue is oxidized to form FGly by a
recently characterized enzyme termed the formylglycine
generating enzyme, or FGE.9 FGE recognizes a consensus
(2) Rosen, S. D. Am. J. Pathol. 1999, 155, 1013-1020.
(3) Farzan, M.; Mirzabekov, T.; Kolchinsky, P.; Wyatt, R.; Cayabyab,
M.; Gerard, N. P.; Gerard, C.; Sodroski, J.; Choe, H. Cell 1999, 96, 667-
676.
(4) Goren, M. B.; Brokl, O.; Schaefer, W. B. Infect. Immun. 1974, 9,
150-158.
(5) Chapman, E.; Best, M. D.; Hanson, S. R.; Wong, C. H. Angew. Chem.,
Int. Ed. 2004, 43, 3526-3548.
(6) Hanson, S. R.; Best, M. D.; Wong, C. H. Angew. Chem., Int. Ed.
2004, 43, 5736-5763.
(8) Dierks, T.; Schmidt, B.; von Figura, K. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 11963-11968.
(9) Preusser-Kunze, A.; Mariappan, M.; Schmidt, B.; Gande, S. L.;
Mutenda, K.; Wenzel, D.; von Figura, K.; Dierks, T. J. Biol. Chem. 2005,
280, 14900-14910.
(7) Boltes, I.; Czapinska, H.; Kahnert, A.; von Bulow, R.; Dierks, T.;
Schmidt, B.; von Figura, K.; Kertesz, M. A.; Uson, I. Structure 2001, 9,
483-491.
10.1021/ol052623t CCC: $33.50
© 2006 American Chemical Society
Published on Web 12/09/2005

Scheme 1. Synthesis of Fmoc-FGly Building-Block 8
sequence around the target cysteine that is highly conserved
reductant in this sequence, as other conditions (i.e., DIBAL-
H, NaBH4 in protic media, or LiAlH4) partially cleaved the
Fmoc group. Simultaneous protection of the aldehyde and
ring opening were effected in refluxing EtOH with catalytic
CSA to furnish amino alcohol 7. Finally, TEMPO-mediated
oxidation18 of the primary alcohol provided the desired SPPS
building block 8 in high yield. Notably, all intermediates
were either taken on crude or purified via recrystallization.
across all species and is found in all sulfatases.6 Interestingly,
some bacterial sulfatases employ a serine residue in place
of cysteine while retaining the overall consensus sequence.
These sulfatases are modified by a distinct mechanism.10
Biochemical studies of sulfatases and FGEs could benefit
tremendously from the availability of synthetic peptides
bearing the central FGly residue. Currently, one scalemic
synthesis of FGly has been reported, which utilizes the highly
versatile Williams’ glycine template.11 However, an Fmoc-
protected variant has not been reported and FGly analogues
have not been incorporated into synthetic peptides. Here we
report an efficient synthesis of a Fmoc-protected FGly
building block and its incorporation into a synthetic peptide
derived from a Mycobacterium tuberculosis sulfatase. The
procedure requires no chromatography and produces Fmoc-
FGly in 6 steps in >70% overall yield.
With building block 8 (Fmoc-FGly(OEt)2-OH) in hand,
we sought to determine its enantiomeric purity and coupling
efficiency for incorporation into peptides. Thus, the FGly
building block was coupled to both chiral and racemic
R-methylbenzylamine with use of EDC/HOBt.19 Following
extractive workup, 1H NMR analysis of the products formed
from the chiral amine indicated that a single stereoisomer
was formed. Analysis of the reaction containing racemic
amine indicated two diasteriomers were formed in approxi-
mately equal amounts.19 These results suggest that the coupling
reaction proceeds without racemization at the R-carbon, and
that the starting material was g95% enantiomerically pure.
In designing a FGly analogue suitable for solid-phase
peptide synthesis (SPPS), we chose to protect the aldehyde
functionality as an acetal that could be removed alongside
other side chain protecting groups during the acidic cleavage
step of Fmoc-based SPPS. The diethyl variant was utilized
as both literature precedent12 and our own experience
indicated that the dimethyl acetal was too resistant to acidic
cleavage. The synthesis began with protection of com-
mercially available D-Ser-OMe‚HCl, using Fmoc-OSu to
provide Fmoc-protected amino acid ester 413,14 in nearly
quantitative yield (Scheme 1). Exposure to catalytic BF3‚
Et2O and 2,2-dimethoxypropane afforded oxazolidine 5 in
excellent yield.15 Sequential reduction and TEMPO-mediated
oxidation16 yielded an Fmoc variant of Garner’s aldehyde
With the coupling reaction characterized, we synthesized
the consensus sequence derived from a Mycobacterium
tuberculosis sulfatase, tetradecapeptide 9 (LFGlyTPSRGSLFT-
GRK). Given the sensitive nature of the aldehyde functional-
ity, we expected that standard peptide cleavage cocktails
containing silanes or thiols would not be compatible. Indeed,
exposure of resin-bound model peptides to cleavage cocktails
containing silanes (i.e., TIS or TES) resulted in reduction of
FGly to serine, while addition of ethanedithiol resulted in
quantitative formation of the dithioacetal. Fortunately, thio-
anisole and anisole were found to be satisfactory alternative
scavengers. In addition to these anticipated issues, an
unexpected problem arose when the full-length peptide was
17
(6) in 92% yield over two steps. LiBH4 is the optimal
(10) Szameit, C.; Miech, C.; Balleininger, M.; Schmidt, B.; von Figura,
K.; Dierks, T. J. Biol. Chem. 1999, 274, 15375-15381.
(11) DeMong, D. E.; Williams, R. M. Tetrahedon Lett. 2002, 43, 2355-
2357.
(12) DeMong, D. E.; Williams, R. M. J. Am. Chem. Soc. 2003, 125,
8561-8565.
(13) While the L isomer of 4 is commercially available and has been
made synthetically (see ref 14), for cost effeciency and safety we adopted
another route.
(15) McKillop, A.; Taylor, R. J. K.; Watson, R. J.; Lewis, N. Synthesis
1994, 31-33.
(16) Janusz, J.; Gryko, D.; Kobrzycka, E.; Gruza, H.; Prokopowicz, P.
Tetrahedon 1998, 54, 6051-6064.
(17) Brown, H. C.; Narasimhan, S.; Choi, Y. M. J. Org. Chem. 1982,
47, 4702-4708.
(18) Zhao, M.; Li, J.; Mano, E.; Song, Z.; Tschaen, D.; Grabowski, E.
J. J.; Reider, P. J. Org. Chem. 1999, 64, 2564-2566.
(19) See the Supporting Information.
(14) Gomez-Vidal, J. A.; Forrester, M. T.; Silverman, R. B. Org. Lett.
2001, 3, 2477-2479.
132
Org. Lett., Vol. 8, No. 1, 2006

cleaved, namely observation of an abundant byproduct
(∼50% by HPLC peak integration) with a mass 172 Da
greater than the desired product. The side reaction occurred
independently of the cleavage cocktail and the abundance
of the byproduct increased as a function of time. Screening
of model tripeptides indicated that the reaction was associated
with the Fmoc-Arg(Pbf)-OH building block. Purification of
the byproduct formed from a model tripeptide (Ac-Arg-FGly-
Ala-OH) allowed its structure to be assigned as 10 (Figure
2) by HRMS and 2D NMR.19
Figure 4. HPLC and mass spectrometry analysis of 9.
of amino acid side chains. These are avoided by the inclusion
of nucleophilic scavengers in the cleavage reaction (i.e.,
silanes, EDT, or anisole). However, the electrophilic nature
of the FGly side chain produces a novel situation in which
the scavengers have either no effect or, worse, react with
the FGly residue. We attempted to avoid the side reaction
during peptide cleavage using (1) various nucleophilic
scavengers, (2) altered cleavage temperatures, or (3) electro-
philic scavengers (i.e., aromatic aldehydes or 1,3-dicarbonyl-
containing compounds). None of these conditions suppressed
formation of the byproduct. We therefore conclude that the
Pbf protecting group is incompatible with FGly.
Figure 2. Modified tripeptide 10.
This unique structure likely results from a Friedel-Crafts-
type reaction between the cleaved 2,2,4,6,7-pentamethyldihy-
drobenzofuran-5-sulfonyl (Pbf) protecting group and FGly
(Figure 3). The initial attack of the electron-rich aromatic
Figure 3. Proposed mechanism for the formation of byproduct.
ring (11) on the FGly aldehyde could occur either before or
after desulfonation to form transient intermediate 12. Loss
of either a proton or SO3 would provide 13, an adduct we
detected by LCMS analysis. Subsequent dehydration then
results in the proposed modified peptide 14.
Figure 5. Mass spectrometry analysis of peptide 9 and its oxime
adduct 15.
This was an interesting development as most side reactions
during peptide cleavage result from the nucleophilic nature
Org. Lett., Vol. 8, No. 1, 2006
133

Accordingly, an alternative arginine building block was
employed. With use of commercially available Fmoc-Arg-
(Boc)2-OH, the synthesis and cleavage of tetradecapeptide
9 was accomplished without incident (Figure 4). Exposure
of purified peptide to MeONH2 afforded complete conversion
of the aldehyde to oxime 15 (Figure 5), confirming that the
aldehyde retains normal reactivity.
In summary, the Fmoc-FGly building block and SPPS
methods for its use that we report here should facilitate
studies of sulfatases and FGEs that activate them. In addition,
the ability to synthesize peptides with internal electrophilic
groups such as the aldehyde may find use in generating cyclic
peptides or in conjugating unnatural epitopes to peptides for
biological studies.
Acknowledgment. We thank Fiona Lin for NMR as-
sistance. This work was supported by a grant to C.R.B. from
the National Institutes of Health (AI51622).
Supporting Information Available: Experimental pro-
cedures, spectral and analytical data, and 2D-NMR spectra
for 10. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL052623T
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Org. Lett., Vol. 8, No. 1, 2006