Synthesis of the antimicrobial S-Linked glycopeptide, glycocin F

Article abstract of DOI:10.1002/chem.201405692

The first total synthesis of glycocin F, a uniquely diglycosylated antimicrobial peptide bearing a rare S-linked N-acetylglucosamine (GlcNAc) moiety in addition to an O-linked GlcNAc, has been accomplished using a native chemical ligation strategy. The synthetic and naturally occurring peptides were compared by HPLC, mass spectrometry, NMR and CD spectroscopy, and their stability towards chymotrypsin digestion and antimicrobial activity were measured. This is the first comprehensive structural and functional comparison of a naturally occurring glycocin with an active synthetic analogue.

Full text of DOI:10.1002/chem.201405692

DOI: 10.1002/chem.201405692
Communication
&
Glycopeptides
Synthesis of the Antimicrobial S-Linked Glycopeptide, Glycocin F
Margaret A. Brimble,*[a, b] Pat J. Edwards,[c] Paul W. R. Harris,[a, b] Gillian E. Norris,[c]
Mark L. Patchett,[c] Tom H. Wright,[a, b] Sung-Hyun Yang,[a] and Sarah E. Carley[a]
Abstract: The first total synthesis of glycocin F, a uniquely
diglycosylated antimicrobial peptide bearing a rare S-
linked N-acetylglucosamine (GlcNAc) moiety in addition to
an O-linked GlcNAc, has been accomplished using
a native chemical ligation strategy. The synthetic and nat-
urally occurring peptides were compared by HPLC, mass
spectrometry, NMR and CD spectroscopy, and their sta-
bility towards chymotrypsin digestion and antimicrobial
activity were measured. This is the first comprehensive
structural and functional comparison of
a naturally
occurring glycocin with an active synthetic analogue.
Glycosylation is a ubiquitous post-translational modification of
proteins in all domains of life.[1] In Nature, the most common
types of glycosylation involve a covalent linkage between
a sugar and either an asparagine sidechain (N-linked) or
a serine or threonine sidechain (O-linked), whereas S-linked
glycopeptides are very rare.[2] Currently, three bacterial glyco-
peptides containing carbohydrate moieties covalently linked to
the sulfur atom of cysteine residues have been reported;
namely, sublancin 168,[3] glycocin F (1)[4] and thurandacins A
and B[5] (Figure 1). All three are bacteriocins, that is, ribosomally
synthesised antimicrobial peptides that have a narrow phylo-
genetic spectrum of activity.
Figure 1. Structures of S-linked glycopeptides: a) sublancin 168;[3]
b) glycocin F (1);[4] c) thurandacins A and B.[5]
Sublancin 168,[3] produced by Bacillus subtilis 168, contains
a b-S-linked glucose moiety, while glycocin F, produced by
Lactobacillus plantarum KW30, contains two b-linked N-acetyl-
glucosamine (GlcNAc) moieties attached to the sulfur atom of
Cys43 and the g-oxygen of Ser18. Sublancin 168 was initially
synthesized by Hojo et al.[6] using a linear Fmoc solid phase
peptide synthesis (SPPS). More recently, Payne and co-work-
ers[7] utilised Fmoc SPPS with a convergent native chemical
ligation (NCL) approach to obtain sublancin 168 and glycosyl-
ated analogues. In 2014, van der Donk et al.[5] reported the in
vitro reconstitution of the biosynthesis of thurandacins A and
B using recombinant gene products of the thuA and thuS
genes from a putative bacteriocin gene cluster in Bacillus thur-
ingiensis serovar andalousiensis BGSC 4AW1.[4] Thurandacins A
and B both have an S-linked glucose moiety; thurandacin B
also contains a b-O-linked glucose unit.[5]
Glycocin F is the only glycosylated bacteriocin which has
been shown to be “glycoactive”, that is, the GlcNAcs are
essential for activity. The enhanced chemical and biochemical
stability of S-glycosidic bonds may provide a rationale for the
existence of these rare linkages in bacteriocins. For example,
they may be resistant to the glycosyl hydrolase repertoire of
producer and/or target organisms,[8] thus contributing to the
expanding area of glycoproteins as therapeutics.[9]
[a] Prof. M. A. Brimble, Dr. P. W. R. Harris, T. H. Wright, Dr. S.-H. Yang,
Dr. S. E. Carley
School of Chemical Sciences, The University of Auckland
23 Symonds St., Auckland 1142 (New Zealand)
E-mail: m.brimble@auckland.ac.nz
[b] Prof. M. A. Brimble, Dr. P. W. R. Harris, T. H. Wright
Maurice Wilkins Centre for Molecular Biodiscovery
School of Biological Sciences, The University of Auckland
Auckland 1142 (New Zealand)
The discovery of glycocin F (1) has provided a unique oppor-
tunity to investigate the functional significance of S-linked
glycosides. Glycocin F is comprised of two a-helices tethered
to each other by two nested disulfide bonds at Cys5/Cys28
and Cys12/Cys21, and connected by a short loop that presents
[c] Dr. P. J. Edwards, Dr. G. E. Norris, Dr. M. L. Patchett
Institute of Fundamental Sciences, Massey University
Palmerston North 4442 (New Zealand)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201405692.
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the O-glycosylated Ser18, whilst the S-linked sugar at Cys43
presents at the end of a long highly flexible C-terminal tail.[8]
We have focused our efforts on the synthesis of 1 and
herein report its total synthesis utilising a native chemical liga-
tion strategy. The synthesis represents the first example of the
incorporation of a b-S-linked GlcNAc residue into Fmoc SPPS
used in combination with NCL to assemble the full protein.
Given the high content of regularly placed cysteine residues
in the peptide backbone of 1, an NCL[10] strategy was feasible1.
In order to maximise ligation efficiency, we identified two
key ligation sites (Figure 2), taking advantage of the known re-
activity of histidine and methionine thioesters.[11] Glycocin F (1)
was therefore envisioned to be assembled from three frag-
ments: Lys1–Met11, Cys12–His27 bearing the appropriate O-
linked glycoside, and Cys28–Cys43 bearing the corresponding
S-linked glycoside.
Figure 2. Proposed disconnection sites of glycocin F (1).
At the outset it was necessary
to prepare O-glycosylated amino
acid 8 and S-glycosylated amino
acid 9 (Scheme 1), both of which
possess GlcNAc moieties joined
via b-glycosidic linkages to Ser18
and Cys43, respectively. The gly-
cosides were prepared as Na-
Fmoc, O-acetyl-protected build-
ing blocks[12] ready for incorpora-
tion into the peptide sequence
using Fmoc SPPS.
To this end, the synthesis of
O-glycoside 8 was achieved by
[13]
HgBr2
mediated Koenigs–
Knorr glycosylation of protected
serine 6,[14] derived from com-
mercially available l-serine in
two steps, and chloroglycosyl
donor 4[15] in a single step with
concomitant removal of the tert-
butyl ester protecting group.
Pleasingly, the desired b-glycosi-
Scheme 1. Synthesis of O-glycoside 8 and S-glycoside 9. TBAHS=tetrabutylammonium hydrogen sulfate, TFA=-
trifluoroacetic acid.
dic linkage was the major product by NMR analysis. To access
S-glycoside 9,[7] 1-thiosugar 5[16] was treated with protected b-
bromoalanine 7[17] with gentle heating in a biphasic solvent
system using tetrabutylammonium hydrogen sulfate as
a phase-transfer catalyst. Following acidolytic cleavage of tert-
butyl ester using trifluoroacetic acid (TFA) S-glycoside 9 was
provided in high yield (95%).
synthesis of peptide Cys28–Cys43 was successfully accom-
plished using a Rink amide linker immobilised on resin fol-
lowed by elongation of the peptide chain using microwave-as-
sisted Fmoc SPPS (Scheme 2). Fmoc-Cys(GlcNAc(OAc)3) was
coupled using HATU/HOAt and 2,2,6,6-tetramethylpyridine
(TMP) to minimise epimerisation,[19] and a Ser35–Ser36 pseudo-
proline dipeptide[20] was introduced to improve the synthetic
efficiency. Peptide Cys28–Cys43 was obtained following resin
cleavage using cocktail A (94% TFA, 2.5% DODT, 2.5% H2O,
1% triisopropylsilane) and HPLC purification (21% isolated
yield).
With glycosylated building blocks in hand, attention was
turned to the chemical synthesis of the polypeptide building
blocks. To expedite the synthesis of the Cys28–Cys43 fragment,
the C-terminal acid Cys bearing the S-linked N-acetylglucosa-
mine, was mutated to a C-terminal amide to circumvent dehy-
droalanine by-product formation.[18] An alternative strategy
using 2-chlorotrityl-type resins, which would deliver a C-termi-
nal acid, was not considered as they are difficult to load. The
Fragment 10, Thz12-His27-COSBn (Thz=thiazolidine), could
not be accessed using Fmoc-based thioester chemistry;
attempts using both Dawson[21] and Liu[22] protocols failed in
the initial stages of linear peptide synthesis. Therefore, direct
thioesterification of the C-terminal carboxylic acid of the fully
protected peptide, Thz12-His27-OH, was envisaged. According-
ly, aminomethyl resin was functionalised with the hyperacid-
1
A linear Fmoc strategy was initially investigated; this proved unsuccessful
with the generation of complex mixtures.
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Scheme 2. Synthesis of peptide fragments 10, 11, 13 and polypeptides 12 and 14 by native chemical ligation. Oxidative folding of polypeptide 14 to effect
the synthesis of GccF-NH2, 2. a) Iterative Fmoc-SPPS; i) deprotection of Na-Fmoc group: 20% piperidine/DMF (v/v), 758C, 50 W, 3 minꢁ2; ii) coupling of Fmoc-
AA-OH: Fmoc-AA-OH (5 equiv), HATU (4.75 equiv), DIPEA (6 equiv), 758C, 25 W, 5 min, for His and Cys, 478C, 25 W, 5 min; iii) cleavage of peptidyl resin: cock-
tail A [TFA/DODT/H2O/triisopropylsilane (94:2.5:2.5:1, v/v/v/v)] for peptide 11 and cocktail B [TFA/ethandithiol/H2O/triisopropylsilane (94:2.5:2.5:1, v/v/v/v)] for
peptide 10; SS represents Fmoc-Ser(tBu)-Ser(yMe,Mepro)-OH. HATU=N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethan-
aminium hexafluorophosphate N-oxide, HOAt=1-hydroxy-7-azabenzotriazole, TMP=2,2,6,6-tetramethylpyridine, Gn·HCl=guanidinium hydrochloride,
MPAA=4-mercaptophenylacetic acid, TCEP=tris(2-carboxyethyl)phosphine, Thz=thiazolidine, HEPES=4-(2-hydroxyethyl)-1-piperazine-1-ethanesulfonic
acid, EDTA=ethylenediaminetetraacetic acid, TFA=trifluoroacetic acid, DODT=2,2’-(ethylenedioxy)diethane thiol.
labile HMPB linker and the ensuing resin-bound hydroxymethyl
group was esterified with Fmoc-His(Trt)-OH using DIC and cat-
alytic DMAP to prevent epimerisation of His.[23] Elongation was
accomplished using microwave-assisted Fmoc SPPS with
HATU/DIPEA, and Fmoc-Ser(GlcNAc(OAc)3) was coupled as de-
scribed for Fmoc-Cys(GlcNAc(OAc)3). The fully protected pep-
tide was then released from the HMPB linker using 1% TFA in
CH2Cl2, neutralised with DIPEA and treated in situ with benzyl-
mercaptan using DIC/6-Cl-HOBt.[24] Deprotection of sidechain
protecting groups using cocktail B[25] (94% TFA, 2.5% EDT,
revealed only a single predominant peak (see the Supporting
Information) and subsequent purification afforded Thz12-
Ser18(glycoside)-His27-COSBn 10 in 21% yield.
Fragments 10 and 11 were then coupled under NCL condi-
tions [phosphate buffer containing guanidine hydrochloride
(Gn·HCl) and tris(2-carboxyethyl)phosphine·HCl (TCEP)][26] at
pH 7 with 4-mercaptophenylacetic acid (MPAA) as additive to
afford polypeptide Thz12-Ser18(GlcNAc(OAc)3)-Cys43(GlcNAc-
(OAc)3)-NH2 within 4 h, as determined by LC-MS. Unexpectedly,
a 1:1 ratio of two ligation products, both of which exhibited
identical mass spectra, were obtained. Due to the stereospecif-
ic nature of NCL[27] that proceeds with stereochemical integrity,
we hypothesized that complete racemization may have taken
2.5% H2O, 1% triisopropylsilane) gave thioester 10 ([M+2H]2+
,
calcd 1089.49, found 1089.20 Da). Slow gradient HPLC analysis,
used to determine the extent of racemisation at His27,
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place at the problematic histidine C terminus of fragment 10
during activation of the C-terminal carboxylic acid and installa-
tion of the thioester functionality.[23,28] The diastereoisomeric
mixture of ligation products was subjected to unmasking of
Thz to Cys using methoxyamine·HCl at pH 4 to afford Cys12-
Ser18(GlcNAc(OAc)3)-Cys43(GlcNAc(OAc)3)-NH2 12. Subsequent
HPLC purification allowed separation of the diastereomers 12a
and 12b in 16.6 and 17.5% yield, respectively. As we were
unable to unequivocally assign
(5200.5325 Da; see the Supporting Information). Purification by
HPLC yielded GccF-NH2 2a (0.65 mg, 14%) and 2b (0.81 mg,
17%), over two steps.
Having successfully prepared GccF-NH2 glycoproteins 2a
and 2b, epimeric at His27, correct assignment of the natural
product was achieved by direct comparison of the HPLC
retention time with an authentic sample of isolated
glycocin F (Figure 3).
the l- and d-stereochemistry of
the histidine residue in peptides
12a and 12b, both diastereo-
mers were carried forward to
complete the synthesis of the
glycocin F sequence, whereupon
an HPLC comparison could be
made with an authentic sample
of glycocin F.
Accordingly, the epimeric frag-
ments 12a and 12b were treat-
ed separately with peptide 13,
Lys1-Trp4(CHO)-Trp6(CHO)-
Met11-COSCH2CH2-(Lys)5-OH
(prepared by Boc SPPS, see the
Supporting Information) under
NCL conditions. The ligation was
complete within 4 h as judged
by HPLC (see the Supporting In-
formation). Mass spectrometric
analysis of the principal peak in
Figure 3. HPLC chromatograms of: a) GccF-OH 1; b) GccF-NH2 2a; c) GccF-NH2 2b. Inset: observed ESI-MS of the
main peak (calcd mass: 5200.5325; found mass for Lys1-Ser18(GlcNAc(OAc)3)-(d)His27-Cys43(GlcNAc(OAc)3)-NH2
2a: 5200.1736 Da (average isotopes)); observed ESI-MS of the main peak (calcd mass: 5200.5325 Da; found mass
for Lys1-Ser18(GlcNAc(OAc)3)-(l)His27-Cys43(GlcNAc(OAc)3)-NH2 2b: 5200.0655 Da (average isotopes)). Isolated
yield of 14 and 17% over the three steps for GccF-NH2 2a and 2b, respectively, based on protein Lys1-Ser18(Glc-
NAc(OAc)3)-His27-Cys43(GlcNAc(OAc)3)-NH2 2. Chromatographic separations were performed using linear gradient
5-45% B in 40 min, about 1% B per min. Buffer A: H2O containing 0.1% TFA (v/v); buffer B: acetonitrile containing
0.1% TFA (v/v).
both ligations corresponded to
the expected ligation product,
Lys1-Ser18(GlcNAc(OAc)3)-Cy-
s43(GlcNAc(OAc)3)-NH2
([M+4H]4+, calcd 1378.87, found
1379.0 Da for reaction with 12a
and 1379.1 for reaction with
12b). Simultaneous deprotection
of the formyl groups and the O-acetate groups of the separat-
The fact that synthetic GccF-NH2 is modified with a C-termi-
nal amide, in contrast to the free carboxylic acid in the natural
product, should not change its chromatographic behaviour
under the acidic conditions used in RP-HPLC. Furthermore, co-
injection revealed that one epimer 2b co-eluted with natural
glycocin F 1. Epimer 2b was therefore assigned the l-configu-
ration at His27 while epimer 2a eluted faster and was assigned
the d-configuration (see the Supporting Information). Diges-
tion of synthetic glycocin F 2b by chymotrypsin which cleaves
primarily at Phe, Tyr and Trp residues, and analysis of the resul-
tant peptide fragments, confirmed the disulfide arrangement
to be identical to native glycocin F (see the Supporting Infor-
mation). Similarly, the CD spectra (Figure 4) showed that both
the synthetic and natural product peptides exhibit the features
expected of a-helical proteins, namely standard double
negative elipticity maxima at 210 and 221 nm, and a positive
maximum near 194 nm.
ed epimeric fragments (epimeric at the histidine residue) was
accomplished using a mixture of hydrazine/2-mercaptoethanol
[7]
(25:37.5, v/v) in NMP/Gn·HCl/HEPES (22.5:5.6:9.4, v/v/v)
to
give polypeptide 14 (Scheme 2). To circumvent loss of material,
the crude products were isolated by solid phase extraction
prior to the oxidative folding.
The isolated epimeric glycopeptides 14a and 14b were indi-
vidually subjected to several different folding conditions (see
the Supporting Information); optimal conditions involved use
of a redox couple containing 2 mm cysteine, 0.25 mm cystine
and 0.1 mm EDTA in 1.5m Gn·HCl (pH 8.2)[29] at 48C which af-
forded a single folded species from each epimeric glycopep-
tide 14a or 14b, respectively, as determined by HPLC, within
16 h. The presence of two disulfide bonds was confirmed by
high resolution mass spectrometry (HRMS) with an observed
mass for synthetic glycocin F-NH2 2a and 2b, epimeric at
His27, of 5200.1736 and 5200.0655 Da, respectively, which is
consistent with the calculated mass of the desired product
The IC50 values of the native and both synthetic glycocins
against Lactobacillus plantarum ATCC 8014 were measured
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Figure 4. Far-UV CD spectra of Cp-5. Blue line: CD spectrum of the synthetic
glycocin F-NH2; red line: CD spectrum of the native glycocin F-OH.
Figure 6. The effect of free GlcNAc on GccF-mediated growth inhibition.
GlcNAc (5 mm) was added to L. plantarum ATCC 8014 cultures at 100 or
200 min after the addition of 8 nm natural product GccF 1, 75 nm 2a, or
25 nm 2b at 0 min.
using liquid culture assays (see the Supporting Information).
Native GccF has an IC50 of 0.9 nm, while that of 2b and 2a are
1.6 and 4.8 nm, respectively (Figure 5).
The natural product is therefore approximately 1.8 times
more active than the l-epimer of the synthesised peptide, and
5.3 times more active than the d-epimer. Not only is the syn-
thetic molecule active against L. plantarum ATCC 8014, but its
effect is attenuated by the addition of free GlcNAc, implying
that it is inhibiting cells using the same mechanism (Figure 6).
The slightly lower antimicrobial activity of 2b may be due to
amidation of the C terminus—the only obvious chemical differ-
ence between the native and synthetic protein. It is known
that the C terminus is a major contributor to activity. As the
assay was conducted at pH 5.6, the loss of a negative charge
at this terminus could affect GccF-target interactions. The l-
isomer at His27 of syn-GccF (compound 2b) is expected to
have the same conformation as the natural product. HSQC
data were collected on 2b under the same conditions as those
used to characterise the natural product.[8] The NMR spectrum
of the synthetic material 2b is consistent with the structure for
the native peptide, bearing in mind the slight localised
conformational differences (see the Supporting Information).
In summary, the first total synthesis of glycocin F, containing
both S- and O-linked GlcNAcs, was accomplished using
a native chemical ligation strategy. Structural similarity to the
natural product was confirmed by HPLC, NMR, CD and chymo-
trypsin digestion. To our knowledge, this is the first chemically
synthesised glycocin that has been shown to possess
biological activity.
Acknowledgements
We thank Mr. Trevor Loo (Massey University) for purifying
natural glycocin F and performing the CD and chymotrypsin
digestion analyses.
Keywords: antimicrobial peptides · disulfide · glycocin F ·
glycopeptides · glycosylation
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Received: October 21, 2014
Published online on && &&, 0000
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Chem. Eur. J. 2015, 21, 1 – 7
www.chemeurj.org
6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Communication
COMMUNICATION
S into Sugar: The first total synthesis of
antimicrobial peptide glycocin F has
been accomplished; subsequent biolog-
ical evaluation showed that synthetic
glycocin F exhibits antimicrobial activity.
Glycocin F exhibits a rare S-linked
N-acetylglucosamine (GlcNAc) moiety in
addition to an O-linked GlcNAc and was
synthesised using a native chemical
ligation strategy.
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Glycopeptides
M. A. Brimble,* P. J. Edwards,
P. W. R. Harris, G. E. Norris, M. L. Patchett,
T. H. Wright, S.-H. Yang, S. E. Carley
&& – &&
Synthesis of the Antimicrobial S-
Linked Glycopeptide, Glycocin F
Chem. Eur. J. 2015, 21, 1 – 7
www.chemeurj.org
7
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ