Selenenylsulfide-linked homogeneous glycopeptides and glycoproteins: Synthesis of human "hepatic Se MetaboliteA"

Article abstract of DOI:10.1002/anie.201106658

Introducing selenium: The synthesis and full characterization of human hepatic Se metaboliteA has been accomplished by using a robust, efficient, and Cys-specific selenenylation protocol (see scheme; NaPi=sodium phosphate). The selenenylsulfide linkage is sufficiently stable to allow quantification of Se-containing glycoconjugates in biological fluids by using atomic detection methods. Copyright

Full text of DOI:10.1002/anie.201106658

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DOI: 10.1002/anie.201106658
Metabolites
Selenenylsulfide-Linked Homogeneous Glycopeptides and
Glycoproteins: Synthesis of Human “Hepatic Se Metabolite A”**
Omar Boutureira, GonÅalo J. L. Bernardes, Marta Fernꢀndez-Gonzꢀlez, Daniel C. Anthony,
and Benjamin G. Davis*
Selenium is a critical mammalian trace element found in
proteins in the form of selenocysteine (SeCys) or selenome-
thionine (SeMet), but its role and metabolism are only
partially understood.[1–3] Early studies[1,2] have surprisingly
shown excretion of selenium as selenosugar 1, so-called
“hepatic Se metabolite B”; its presence in rats and humans
(liver and urine) also suggests a common cross-species
excretion mechanism (Scheme 1). These studies implicated
4, so-called “hepatic Se metabolite A”, which was neither
isolated in pure form nor fully characterized, as the precursor
on the basis of MS fragmentation. Selenosugars 2 and 3 have
also been detected in urine, but, notably, glutathionyl
precursors (e.g., 5) of these trace metabolites have not yet
been identified.[3] Indeed, the exact mechanism of selenium
metabolism remains unclear, as do the roles of 4 and 5, and
the need for authentic standards has recently been empha-
sized.[4]
The putative biological roles as well as the intrinsic
characteristics of selenium make it potentially useful as a
structural, functional, and mechanistic probe for the detailed
study of biological processes by 77Se NMR[5–7] and X-ray
spectroscopy,[8] and a tool for the determination of phase
through the use of multiwavelength anomalous dispersion
(MAD).[9] Because selenium is not present in detectable
levels in the background and can be quantified at sub-mm
concentrations, it also potentially allows sensitive estima-
tion[10–12] of in vivo circulation lifetime and tissue distribution
Scheme 1. Putative pathway for the biosynthesis of selenium-contain-
ing natural products 1–3. Methyltransferase activity has been inferred
through inhibition studies.[2] OH epimerase in mammalian cells[13]
interconverts GlcNAcUDP and GalNAcUDP, but is unknown for the
other substrates shown here.
of seleno conjugates.
Selenenylsulfide-linked (glycosyl-SeS-Cys) glycopeptides,
which form a class of conjugates that has not been synthesized
previously, are therefore not only of fundamental interest as
[*] Dr. O. Boutureira, Dr. G. J. L. Bernardes,
Dr. M. Fernꢀndez-Gonzꢀlez, Prof. B. G. Davis
Department of Chemistry, University of Oxford
Chemistry Research Laboratory
human metabolite natural products that might allow dissec-
tion of selenium utilization pathways, but also suggest
potential as readily monitored labelled glycoconjugates.
Herein, we present the first syntheses of such conjugates,
including the identification of hepatic Se metabolite A, the
creation of protein-conjugate variants, and an analysis of their
serum-tracking potential and stability.
Mansfield Road, OX1 3TA (UK)
E-mail: ben.davis@chem.ox.ac.uk
Homepage: http://users.ox.ac.uk/~dplb0149
Dr. D. C. Anthony
Department of Pharmacology, University of Oxford
Mansfield Road, Oxford, OX1 3QT (UK)
Sugar selenenylsulfides are rare and few syntheses toward
them have been reported to date;[14] sugar selenenylsulfides
[**] We thank the European Commission (Marie Curie Intra-European
Fellowship for O.B.), FCT, Portugal (G.J.L.B.), and Xunta de Galicia,
Spain (M.F.G.) for financial support. B.G.D. is a recipient of a Royal
Society Wolfson Merit Award and is supported by an EPSRC LSI
Platform grant. We thank Dr. J.S.O. McCullagh and M.K. Patel for
technical assistance.
À
À À
with the sugar Se S R constitution that is found in target
compounds 4 and 5 are not known to date, and it has indeed
been suggested that such compounds may be too unstable to
be isolated.[2] We investigated the synthesis of hepatic Se
metabolite A (4) and the related glutathionyl 5 (the putative
precursor to 3), using a method that exploited the high
reactivity of diselenides in thiol–diselenide exchange[14–16]
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201106658.
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reactions with cysteine (Scheme 2).[17] Notably, the relative
stability of diselenides does not lead to a reduced reactivity,[18]
and we previously noted that kinetic control may be applied
resonances. In particular, diagnostic signals of diastereotopic
protons corresponding to Cys-CH2 appeared in the H NMR
1
spectrum at 3.37 ppm (JA,B = 14.3 Hz, JA,Ha = 4.8 Hz) and
3.13 ppm (JA,B = 14.3 Hz, JB,Ha = 9.5 Hz), and in the
13C NMR spectrum at 39.1 ppm (CbH2SSe). ESI-MS spectra
showed the expected isotope distribution that is characteristic
for Se also for the molecular ion. ESI-MS/MS measurements
allowed fragmentation analysis of the synthetic GalNAc-SeS-
G (4), which showed essentially identical patterns for both
molecular (including isotopic distribution) and fragment ions
to those reported previously[2] (see the Supporting Informa-
tion).
The GlcNAc 4-OH epimer 5 was targeted next, both to
investigate its role as a putative precursor of 3 and to test
other possible configurations for hepatic Se metabolite A.
The synthesis of 5 proved more challenging; direct monitoring
of the thiol–diselenide exchange (see the Supporting Infor-
mation) indicated complete conversion of GSH and
(GlcNAcSe)2 (6b) to expected product 5 after 30–45 min.
However, compound 5 proved to be significantly more labile
than compound 4. Only traces of 5·TFA were detected after
purification by reverse-phase (RP) HPLC. Purification by
size-exclusion chromatography afforded recovered diselenide
6b (56%) along with a second, mixed fraction containing 6b/
5/(GS)2 in a ratio of 3:1:3, as determined by 1H NMR
spectroscopy. The presence of the three different dichalcoge-
nide moieties (Se2, SeS, and S2) in the same fraction can be
Scheme 2. Synthesis of putative Se-metabolism precursors 4 and 5.
DMF=N,N-dimethylformamide, py=pyridine.
to such reaction manifolds. Although we had exploited this to
favor selective mixed-disulfide formation,[14] we reasoned that
the reaction might also allow access to selenenylsulfide-linked
intermediates if correct conditions could be found for these to
be trapped.[19] Sugar diselenides 6a–f were prepared in two
steps from the corresponding anomeric halides in good
overall yields[20] (see Scheme 2 for 6a,b, and the Supporting
Information for 6c–f). Conditions for the reaction of dis-
elenides with l-glutathione (GSH) were tested by varying
several parameters, but were limited where possible to those
in aqueous biologically compatible buffers at a nearly neutral
pH value, so that the reaction might then be applied to the
investigation of other more complex SeS-linked glycoconju-
gates (Scheme 2). The optimization of reaction conditions
was facilitated by direct monitoring of the reaction by using
MS (negative mode TOF-ES). Signals that indicated the
conversion of GSH (m/z 306) to product 4 (m/z 589) and a
minor signal attributed to the formation of (GS)2 (m/z 611)
were monitored directly; the latter signal increased with
longer reaction times (1–3 h, see the Supporting Information).
Thus, reaction of GSH with (GalNAcSe)2 6a in sodium
phosphate (NaPi) buffer (50 mm, pH 8.0) at room temper-
ature for 30 min afforded hepatic Se metabolite A (GalNAc-
SeS-G, 4) in 62% yield (based on the limiting peptide, > 95%
conversion, see the Supporting Information); 4 was isolated
both in its pure form and as trifluoroacetate salt 4·TFA with
GS2 by using size-exclusion chromatography and RP-HPLC,
respectively (see the Supporting Information). Notably,
kinetic control of the thiol–diselenide exchange is sufficient
to result not only in reasonably efficient formation of the
product, but also in the stereoselective formation of the
À
tentatively attributed to comproportionation of the Se S
bond in 5 under these conditions; mixed samples that were
incubated in D2O at a lower temperature of 48C for 7 days
showed no signs of additional degradation/comproportiona-
tion.[15,21,22] Structural analyses of the product mixtures by
mass spectrometry and NMR spectroscopy allowed charac-
terization of putative glutathionyl precursor GlcNAc-SeS-G
(5) as its b anomer (d.r. > 98%, 1JH1–C1 = 159.1 Hz, see the
Supporting Information). The structure of 5 was confirmed
also by HRMS and a distinct characteristic isotope pattern for
the molecular ion in ESI-MS. Importantly, ESI-MS/MS
analyses showed different fragmentation patterns for 4 and
5. The trends in the dependence of the fragmentation
behavior on both collision energy and configuration were
also consistent with those observed by Ogra et al.[23] for
various selenomethyl N-acetylhexosamines (including those
of GalNAc and GlcNAc).
Having gained valuable insight into reactivity and stability
of sugar-SeS-G conjugates from their investigation under
biologically compatible conditions, we next investigated more
complex polypeptide substrates. It cannot be discounted[2]
that metabolites, such as 1–3, might also arise from methyl-
transferase activity (Scheme 1) both on sugar-SeS-glutathione
conjugates and on sugar-SeS-protein conjugates. We reasoned
that investigation of polypeptide substrates would also test
the generality of a potentially straightforward convergent
site-specific protein-labeling method for the synthesis of SeS-
linked homogeneous glycopeptides and glycoproteins
(Scheme 3). If successful, this method would allow the
ready combination of flexible site-specific glycoconjuga-
tion[24,25] with efficient chemical incorporation of selenium
into proteins as an additional tag-and-modify[26,27] strategy.
1
b anomer (d.r. > 98%, JH1–C1 = 159.7 Hz), thus out-compet-
ing any glycosylselenyl mutarotation. The product was
characterized by 1D and 2D (COSY, HSQC) 1H and
13C NMR experiments, thus allowing full assignment of all
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Table 1: Site-specific syntheses of homogeneous SeS-linked glycopro-
teins 8a–f.[a]
Entry Selenenylating
Reagent
t [h] Product
Conv. [%]
ESI-MS
(Sugar, R)
Found (Calcd)
8a
>95
1[b]
2[b]
3
6a
6b
6c
6d
6e
6 f
2
(Gal, NHAc) 26998 (26998)
Scheme 3. General strategy for the chemical site-specific incorporation
of selenoglycosides into proteins/peptides with different groups at the
C-2 position in the carbohydrate moiety.
8b
>95
2
1
2
2
2
(Glc, NHAc) 26996 (26998)
Chemical site-specific protein modification is a potentially
powerful method to access homogeneous posttranslationally
modified proteins and to introduce synthetic probes that are
now key tools for understanding complex biological process-
es.[27–29] Selenium can provide the basis for structural deter-
mination and monitoring of protein surfaces and/or active-site
events.[30–33] Most methods for the incorporation of selenium
into proteins use the biosynthetic cell machinery.[34–40] Chem-
ical methods (beyond the limits of SeCys and SeMet) have
also been exploited as an alternative to genetic techniques,
but to date the reported methods suffer from low efficiency
(2–50%) and are restricted to active-site residues of pro-
teins.[41] Peptide modifications are mainly focused on solid-
phase techniques in combination with native-chemical-liga-
tion strategies.[30,41–47] We envisioned the glycoselenenylation
method developed in this work to be possibly valuable as it
would allow the simultaneous incorporation of glycans, which
is a widespread type of protein modification.[48]
8c
(Glc, OH)
>95
26955 (26956)
8d
(Glc, F)
>95
26960 (26958)
4
8e
(Man, F)
>95
26959 (26958)
5
8 f
(Gal, F)
>95
26960 (26958)
6
[a] Reactions carried out at 48C with 100 equiv of sugar diselenide.
[b] Reactions carried out at RT; no product formation at 48C.
nosyl (FDM), and b-d-galactosyl (FDGal) glycoproteins 8d–f
was also achieved with excellent conversions (> 95%;
Table 1, entries 4–6).
A single-cysteine mutant (SBL-S156C, 7) of subtilisin
from Bacillus lentus was used as a model protein substrate. To
À
test the glycan breadth of this Se S bond-forming reaction,
The functional aspects of such SeS-linked glycoproteins
were explored next. A key prerequisite is the preservation of
the protein state. Importantly, modified glycoproteins
retained inherent (enzymatic peptidase) activity (see the
Supporting Information). Thus, the selenenylating method
allows attachment of different potential probes (Se and
glycan) without compromising protein function. Moreover,
we reacted 7 with a range of reagents, which were prepared by
using essentially analogous routes to those shown in Scheme 2
(see the Supporting Information and Table 1). Thus, thiol–
diselenide exchange was successfully carried out by simply
mixing sugar diselenide reagents 6a–c with 7 in phosphate
buffer at pH 8.0; the reactions proceeded to essentially full
conversion within 30 min–2 h (Table 1, entries 1–3). The
formation and isolation of these sugar-SeS-protein conju-
gates, which contain natural sugars that are known to be
processed by glycosyltransferases (Gal, GlcNAc, and
GalNAc), suggest that, at least in principle, such protein
adducts would also be potentially viable intermediates in
equivalent pathways to that shown in Scheme 1 for 4 and 5.
To explore the scope of this method, we decided to apply
the conditions described above to the preparation of glyco-
conjugates that bear different sugars, including non-natural
derivatives, such as 2-fluoro-substituted analogues (Table 1,
entries 4–6). There is a growing interest in the synthesis of
fluorinated carbohydrates and their corresponding fluoro-
substituted glycoconjugates.[49–52] The synthesis of SeS-linked
2-deoxy-2-fluoro-substituted b-d-glucosyl (FDG), b-d-man-
À
the stability of the newly formed Se S bond was sufficient to
permit the subsequent modification to more complex glycans
through endoglycosidase-catalyzed transglycosylation.[53]
The ready introduction of a selenium tag into proteins has
the potential to be advantageously employed for the detec-
tion, quantification, and tracking, for example during biodis-
tribution studies by atomic detection methods. When com-
pared to, for example, more standard ELISA or radiolabeling
methods, such methods have advantages, including low
detection limits, absence of radiation hazards, and the ability
to distinguish between exogenous and endogenous seleno-
proteins by using different Se isotopes. We were delighted to
find that selenoglycoprotein SBL-C156-SSe-Glc 8c could be
detected at a detection limit of as low as 20 ppb, even when
greatly diluted in rat plasma, by using inductively coupled
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plasma atomic emission spectrometry (ICP-AES), thus pro-
viding an important proof-of-principle for the use of such
selenoconjugates as putative biological probes.
As well as acting as possible probes of glycoprotein
function, we believe such sugar-SeS-R conjugates might shed
further light on the reactivity, stability, and metabolism of
selenosugar. Our full data for GalNAc-SeS-G 4 are consistent
with the partial data obtained previously for the isolated
natural product hepatic Se metabolite A[2] and suggest that
the previously proposed identity is correct. Moreover, the
stabilities of 4 and 5 vary dramatically according to the
configuration of the attached sugar. Although unexpected,
this result allows us to propose an explanation for the
observation that the more unusual glycosyl moiety GalNAc is
the dominant sugar product of this human biosynthetic
pathway. Regardless of the exact mechanism and the path-
way(s) of its formation, GalNAc compound 4 is stable under
conditions (aqueous and plasma, see above) under which
GlcNAc 5 is not stable. These observations are consistent with
prior suggestions that the stability of the SeS linkage is
influenced by stereoelectronics and linkage environment (as
well as by the pH value of the medium).[55] The difference in
the stability of 4 and 5 mirrors the eventual proportions of Se
metabolites that are found in human urine: the concentration
of minor GlcNAc Se metabolite 3 is less than 2% of the
concentration of GalNAc variant 1.[58] 4 and 5 as well as 1 and
3 are pairs of 4-OH epimers; 4-OH sugar epimerase activity,
which interconverts GalNAc with GlcNAc derivatives, is
prominent in mammalian cells.[13] It may be that promiscuous
methyltransferase activity (Scheme 1) processes available
substrates and, from our results gathered here, is likely to
favor GalNAc-SeS-R as a precursor on the basis of stability.
These putative biosynthetic intermediates 4 and 5 will there-
fore be valuable in ongoing studies to isolate the associated
human glycosyl- and methyl-transferases.
We next investigated the stability of the conjugate, with a
view especially to potential in vivo applications. Moreover,
we reasoned that the relative stability of SeS-linked inter-
mediates might play a role in the suggested biosynthetic
pathway outlined in Scheme 1. Notably, the higher stability of
selenenylsulfide bonds as compared to disulfides has been
exploited for solving peptide-folding problems and in protein
engineering;[54,55] in addition to the favorable formation of
SeS over S2,[56] the former possesses a lower redox potential
(typically 70 mV lower than the corresponding disulfides).[19]
Glutathionyl-sugar conjugates 4 and 5, and glycoprotein SBL-
C156-SSe-Glc 8c were incubated in rat plasma. No break-
down of either of the SeS-linked conjugates 4 and 8c was
detected over 30 h at 378C, thus suggesting that both are
resistant and stable in vitro under these conditions (see the
Supporting Information). The stability of SeS-linked glyco-
protein conjugate SBL-C156-SSe-Glc 8c is consistent with
previous observations for BSA-ebselen complexes.[57] Puta-
tive biosynthetic intermediate GalNAc-SeS-G 4 proved
stable even over prolonged periods of up to 95 h at 378C in
rat plasma; in striking contrast, epimer GlcNAc-SeS-G 5
proved insufficiently stable even in aqueous buffer (see
above) and could not be observed in plasma. Notably, all of
the SSe-linked protein conjugates showed good stability
regardless of configuration; the enhanced stability is tenta-
tively attributed to reduced accessibility of the SSe linkage.
In summary, the first target synthesis and full character-
ization of human hepatic Se metabolite A has been accom-
plished by using a robust, efficient, and Cys-specific selene-
nylation protocol, which also allowed the first synthesis of the
putative intermediate GlcNAc-SeS-G 5. Although this
metabolite has not yet been identified, it seems a logical
precursor (Scheme 1) to minor natural product 3 of the
human Se-metabolism pathway. The substrate scope and
Received: September 19, 2011
Published online: December 30, 2011
Keywords: glycoproteins · metabolites ·
posttranslational modifications · selenium · selenoglycosides
.
À
efficiency of this Se S bond-forming method was demon-
strated through its extension to proteins and to several other
glycan types (both natural and non-natural), some of which
allowed simultaneous incorporation of selenium and fluorine
into proteins at predefined sites. To the best of our knowledge,
this strategy also constitutes the first site-specific incorpora-
tion of selenoglycosides into proteins. The integrity of the
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