Synthesis of (2R,3S) 3-amino-4-mercapto-2-butanol, a threonine analogue for covalent inhibition of sortases

Article abstract of DOI:10.1016/j.bmcl.2005.07.073

L-Threonine 2 was converted in seven steps into the protected aminomercaptoalcohol 8, a threonine mimic. This compound 8 was coupled to various oligopeptides to produce two different tetrapeptide analogues, for example, 11 and 17, which were shown to inhibit the Sortase enzymes (SrtA and SrtB) via covalent attachment of the thiol groups of 11 and 17 to the catalytically active cysteine residue of the Sortase enzymes.

Full text of DOI:10.1016/j.bmcl.2005.07.073

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5076–5079
Synthesis of (2R,3S) 3-amino-4-mercapto-2-butanol, a
threonine analogue for covalent inhibition of sortases
Michael E. Jung,a,* Jeremy J. Clemens,a Nuttee Suree,a,b Chu Kong Liew,a,b
Rosemarie Pilpa,a,b Dean O. Campbella,b and Robert T. Clubba,b,c
aDepartment of Chemistry and Biochemistry, University of California, Los Angeles, 405 Hilgard Ave, Los Angeles, CA 90095, USA
bUCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles,
405 Hilgard Ave, Los Angeles, CA 90095, USA
cMolecular Biology Institute, University of California, Los Angeles, 405 Hilgard Ave, Los Angeles, CA 90095, USA
Received 15 June 2005; revised 20 July 2005; accepted 25 July 2005
Available online 19 September 2005
Abstract—L-Threonine 2 was converted in seven steps into the protected aminomercaptoalcohol 8, a threonine mimic. This com-
pound 8 was coupled to various oligopeptides to produce two different tetrapeptide analogues, for example, 11 and 17, which were
shown to inhibit the Sortase enzymes (SrtA and SrtB) via covalent attachment of the thiol groups of 11 and 17 to the catalytically
active cysteine residue of the Sortase enzymes.
Ó 2005 Elsevier Ltd. All rights reserved.
Many surface proteins on Gram-positive bacteria are
covalently anchored to the cell wall by sortase enzymes,
a family of novel cysteine transpeptidases.1 The sortase
A (SrtA) protein from Staphylococcus aureus is required
for bacterial virulence and is the best studied member of
this enzyme family. It ÔsortsÕ proteins to the cell wall by
processing a conserved C-terminally located LPXTG
motif, cleaving the threonine–glycine peptide bond,
and attaching the threonine carbonyl to the amine group
of lipid II.2 This lipid–protein intermediate is then incor-
porated into the peptidoglycan via transglycosylation
and transpeptidation reactions of cell wall synthesis.
Sortases are an attractive target for new antibacterial
agents, since they are widely distributed in a diverse
set of pathogens3 where they are frequently required
for virulence.4 In our ongoing investigations of the
structure and function of these enzymes, structures of
the apo-SrtA enzyme have been solved,5 the LPXTG
binding site has been coarsely defined,6 residues critical
for catalysis have been identified,7 and calcium has been
shown to activate SrtA by promoting the closure of an
active site loop that contacts the substrate.8 At present,
it is not known how these enzymes recognize and pro-
cess their sorting signals, which can vary dramatically
in sequence.3 For example, although the distantly relat-
ed sortase B (SrtB) also cleaves the acyl threonine pep-
tide bond, it recognizes a very distinct sorting signal
containing the amino acid sequence NPQTN.4c To aid
in structural studies designed to elucidate the molecular
basis of substrate specificity, we have previously pre-
pared several oligopeptides that contain threonine ana-
logues that covalently bind to the thiol of Cys184.
These compounds, for example, the vinyl nitrile and
vinyl sulfone analogues of threonine, have indeed been
useful, but have not yet yielded modified enzymes that
are sufficiently homogeneous for detailed structural
studies.6b Therefore, we have developed a new threonine
analogue, which can bind covalently to both SrtA and
SrtB by a different mechanism, namely via a disulfide
bond. We report the synthesis of (2R,3S) 3-amino-4-
mercapto-2-butanol in the doubly protected form 8
and its incorporation into the two distinct target tetra-
peptide sequences for SrtA and SrtB, compounds 11
and 17, respectively. We also report the binding data
for these peptide analogues versus the target enzymes.
Keywords: Threonine analogues; Mercaptomethyl derivatives of
threonine; Sortase A inhibition; Sortase B inhibition; Tetrapeptide
analogue synthesis.
Of
the
many
possible
protected
threonine
mercaptomethyl analogues,9 we chose compound 8 in
which the alcohol and thiol functionalities were protect-
ed with silyl groups, since the removal of protecting
*
Corresponding author. Tel.: +1 3108257954; fax: +1 3102063722;
e-mail: jung@chem.ucla.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.073

M. E. Jung et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5076–5079
5077
groups should be mild enough as to cause no racemiza-
Me
Me
TBDPSO
H2N
tion of any peptide bond. Hydrogen sulfide was convert-
ed (Scheme 1) into the tert-butyldiphenylsilyl (TBDPS)
thiolate 1 in two steps in 72% yield.10 L-Threonine 2
was converted via standard methods into the silyl-pro-
tected methyl ester 3 in an unoptimized yield of 37%.
Protection of the amine as the Boc derivative 4 and hy-
dride reduction gave the alcohol 5 in 45% yield. Conver-
sion to the bromide 6 (50%), followed by SN2
displacement using the thiolate 1, gave the bis-silylated
carbamate 7 in 96% yield. Final Boc removal was quite
difficult on account the sensitivity of the TBDPS-protect-
ing groups. Normal acidic conditions removed one or
both of the silyl groups in addition to the Boc group.
Finally, the following set of conditions were found to
work well and reproducibly, namely treatment of 7 with
gaseous HCl in anhydrous ethyl acetate at 23 °C for
20 min to afford the desired bis-silylated amine 8 in
82% yield.11
Me
O
i
Me
N
OH
CbzHN
N
H
O
O
TBDPSS
9
8
Me
TBDPSO
Me
Me
Me
ii
O
H
N
N
CbzHN
N
H
O
O
STBDPS
10
Me
Me
HO
Me
Me
O
H
N
N
CbzHN
N
H
O
O
HS
11
Scheme 2. Reagents and conditions: (i) EDCI, DMAP, HOBt,
CH2Cl2, 0 °C ! 23°C, 2 h, 86%; (ii) TBAF, THF, 23 °C, 3 h; AcOH,
23 °C, 5 min, 70%.
With the bis-protected amine in hand, we studied its
incorporation into the desired tetrapeptide sequences
for SrtA and SrtB. The tripeptide cbz-LPA (leucine-pro-
line-alanine) 9 was prepared in seven steps using stan-
dard solution-phase peptide synthesis methodology
(Scheme 2). Coupling of 8 with the tripeptide 9 using
EDCI and DMAP gave the desired bis-protected SrtA
tetrapeptide analogue 10 in good yield. Final deprotec-
tion of the two silyl groups with fluoride (TBAF) gave
the tetrapeptide analogue 11 in which the carbonyl
group of threonine has been replaced with a mercaptom-
ethyl (CH2SH) unit.
Due to the somewhat higher reactivity of the amino acid
side chains, for example, the amides of the Asn and Gln
groups, in the tetrapeptide analogue of the SrtB se-
quence, a different strategy was used to prepare the SrtB
analogue 17 (Scheme 3). Coupling of the bis-silylated
amine 8 with a-N-Boc glutamine 12 gave the dipeptide
analogue 13 in 61% yield. Removal of the Boc group,
H2N
O
H2N
BocHN
O
O
TBDPSO
H2N
Me
TBDPSO
i
Me
H
NH2
OH
N
Ph
tBu Si S
iii, iv
BocHN
i, ii
HO
OH
TBDPSS
H2S
MeO
HO
K
O
O
STBDPS
Ph
O
Me
8
12
13
L-Threonine, 2
1
H2N
O
NH2
NHBoc
TBDPSO
NH2
O
vi
ii
v
iii
OTBDPS
MeO
OTBDPS
Me
N
H
N
CbzHN
OH
H2N
O
Me
O
Me
O
O
3
4
STBDPS
H2N
15
14
NHBoc
OTBDPS
NHBoc
OTBDPS
viii
O
vii
Br
O
O
TBDPSO
Me
Me
NH2
N
Me
O
H
N
iv
6
5
CbzHN
N
H
O
NHBoc
OTBDPS
NH2
STBDPS
ix
TBDPSS
TBDPSS
OTBDPS
16
Me
Me
H2N
O
7
O
8
HO
NH2
O
Me
Scheme 1. Reagents and conditions: (i) nBuLi, THF, ꢀ78 °C ! 23°C,
30 min, then 23 °C ! ꢀ78 °C, TBDPSCI, 30 min, quant.; (ii) KH,
pentane, 0 °C, 2 h, 72%; (iii) SOCl2, MeOH, 0 °C ! 23 °C, 18 h; (iv)
TBDPSCI, imidazole, CH2Cl2, 0 °C ! 23 °C, 18 h, 37% (two steps);
(v) (Boc)2O, Et3N, MeOH, 18 h, quant.; (vi) LAH, THF, 0 °C, 2 h,
45%; (vii) Et3N, PPh3, CBr4, CH2Cl2, 23 °C, 18 h, 50% (viii) 1, THF,
ꢀ78 °C, 1 h, then 23 °C, 18 h, 96%; (ix) HCl, anhyd EtOAc, 23 °C,
20 min, 82%.
H
N
N
CbzHN
N
H
O
O
HS
17
Scheme 3. Reagents and conditions: (i) PyBOP, DIEA, CH2Cl2, 23 °C,
18 h, 61%; (ii) HCl, anhyd EtOAc, 23 °C, 30 min, 73%; (iii) PyBOP,
DIEA, CH2Cl2, 23 °C, 1 h, 74%; (iv) TBAF, HOAc, 23 °C, 18 h, 54%.

5078
M. E. Jung et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5076–5079
again using the conditions utilized earlier, for example,
HCl in ethyl acetate, gave the amine 14 in 73% yield.
The Cbz-protected dipeptide (Asn-Pro) 15 was prepared
in four steps using standard solution-phase peptide syn-
thesis methodology. Coupling of the dipeptide analogue
14 to 15 using PyBOP and HunigÕs base gave the desired
tetrapeptide analogue 16 in 74% yield. Final removal of
the two silyl groups with fluoride ion gave the SrtB tet-
rapeptide analogue 17 in 54% yield. This analogue again
has the mercaptomethyl (CH2SH) group in place of the
carbonyl unit of threonine.
In summary, we have developed an efficient method for
the synthesis of the bis-protected L-threonine analogue 8
and have used it in the preparation of two tetrapeptide
analogues of the sorting sequence for SrtA and SrtB,
compounds 11 and 17, respectively. These compounds
covalently modified their respective enzymes at reason-
able concentrations. The use of these new covalently
bound sortase analogues for the determination of the
three-dimensional structure of a bound sortase is cur-
rently under study in our laboratories.
To test the viability of these compounds as possible
covalent binders, the SrtA tetrapeptide analogue 11
(5 M excess) was added to SrtA (5 lL of 20 lM solu-
tion) in a pH 8 buffer (50 mM Tris–HCl, 100 mM NaCl)
with and without CaCl2 and incubated at room temper-
ature. Samples were removed periodically and analyzed
using reverse-phase HPLC on a C18 column. After 20 h,
a large new peak appears, while the original peak for
SrtA disappears (Fig. 1). Likewise, when the SrtB tetra-
peptide analogue 17 was added to SrtB under similar
conditions, a new peak again appears in the reverse-
phase HPLC along with the concomitant disappearance
of the original peak due to SrtB.
Acknowledgments
We thank Dr. Robert Damoiseaux at the UCLA Molec-
ular Screening Shared Resource for his assistance. We
also thank the National Institutes of Health for support
via grant AI52217.
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