Synthesis aided structural determination of amyloid-β(1-15) glycopeptides, new biomarkers for Alzheimer's disease

Article abstract of DOI:10.1039/c4cc05085a

Unique tyrosine glycosylated amyloid-β(1-15) glycopeptides were synthesized with well-defined stereochemistry at the glycosidic linkages. Aided by these glycopeptides and tandem mass spectrometry analysis, the naturally existing amyloid-β glycopeptides, isolated from Alzheimer's disease patients, were determined to contain an α-linked N-acetyl galactosamine at the modified tyrosine 10 residue. Glycosylation can significantly impact the properties of amyloid-β as the glycopeptide has much lower affinity for Cu⁺ ions.

Full text of DOI:10.1039/c4cc05085a

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Synthesis aided structural determination of
amyloid-b(1–15) glycopeptides, new biomarkers
for Alzheimer’s disease†
Cite this: Chem. Commun., 2014,
0, 15067
5
Received 3rd July 2014,
Accepted 8th October 2014
a
b
c
b
Peng Wang, Jonas Nilsson, Gunnar Brinkmalm, Goran Larson* and
¨
a
Xuefei Huang*
DOI: 10.1039/c4cc05085a
www.rsc.org/chemcomm
Unique tyrosine glycosylated amyloid-b(1–15) glycopeptides were early AD development. Therefore, the Ab glycopeptides can be
synthesized with well-defined stereochemistry at the glycosidic potential new biomarkers to facilitate early AD diagnosis.
linkages. Aided by these glycopeptides and tandem mass spectrometry
The structures of Ab glycopeptides were analyzed by mass
4
analysis, the naturally existing amyloid-b glycopeptides, isolated from spectrometry (MS). While glycoproteins and glycopeptides with
Alzheimer’s disease patients, were determined to contain an a mammalian origin commonly bear carbohydrate chains on
a-linked N-acetyl galactosamine at the modified tyrosine 10 residue. serines/threonines (O-linked glycans) or on asparagine residues
5,6
Glycosylation can significantly impact the properties of amyloid-b as (N-linked glycans), Ab glycopeptides contain glycosylation on
+
4
the glycopeptide has much lower affinity for Cu ions.
tyrosine 10 (Tyr10). Although tyrosine glycosylations have been
7–9
found with a glucose-a(1-O)Tyr or a galactose-b(1-O)Tyr linkage,
Alzheimer’s disease (AD) is the most common form of dementia, the carbohydrates on Ab glycopeptides were determined to have a
which is characterized by the formation of amyloid plaques core N-acetyl hexosamine (HexNAc) moiety linked to the hydroxyl
composed of aggregates of amyloid-b (Ab) polypeptides. AD is a group of Tyr. The HexNAc was further extended with one hexose
serious concern for modern society as it is estimated that 5.4 million and two or three N-acetylneuraminic acid residues (Neu5Ac). In
Americans had AD resulting in a cumulative cost of approximately analogy with other O-linked glycans, the core HexNAc was
1
2
00 billion dollars in 2013. One of the major challenges in AD proposed to be N-acetyl galactosamine (GalNAc). An ambiguity
treatment is accurate diagnosis at an early stage. Ab and tau proteins in the structure is that the stereochemistry of the HexNAc–Tyr
2
have been widely viewed as biomarkers for AD. However, they are of linkage could not be defined by the LC-MS/MS analysis alone. The
limited utility in diagnosis at the presymptomatic stage. There is an uncertainty in structure has hindered the understanding of its
urgent need to identify AD biomarkers with higher sensitivity and function as well as the efforts in generating glycopeptide specific
3
specificity before cognitive decline appears. Recently, a series of monoclonal antibodies for its detection.
novel Ab glycopeptides, corresponding to the N-terminal fragments
To enable structural determination, herein, we report the
(
Ab 1–X, X ranging from 15 to 20 residues) of Ab, were isolated total synthesis of Ab(1–15) glycopeptides bearing GalNAc on
4
from the cerebrospinal fluid of patients. It was found that these Tyr10 with well-defined stereochemistry, which joins the few
Ab glycopeptides are present in AD patients at markedly higher examples of chemical synthesis of O-glycosyl tyrosine amino
4
10–13
14
levels compared to those from non-AD control patients. Moreover, acid
and glycopeptide reported to date. Aided by the
cognitively impaired patients considered as non-AD patients due to synthetic Ab(1–15) glycopeptides, the HexNAc in the natural
below threshold Ab(1–42) levels actually exhibited Ab glycopeptide glycopeptide from AD patients was determined to be a-linked
profiles similar to those of AD patients, which may have indicated GalNAc through MS. Glycosylation of Tyr10 can significantly
change the properties of the Ab peptide as evident from the
+
much reduced affinity of the glycopeptide for Cu ions.
a
Department of Chemistry, Michigan State University, 578 S. Shaw Lane,
To access the Ab glycopeptide, we began with the preparation
East Lansing, Michigan 48824, USA. E-mail: xuefei@chemistry.msu.edu
b
of the glycosylated Tyr building block from the D-galactosamine
hydrochloride 1. As stereochemistry at the glycosyl linkage is not
known, we aimed for both a and b anomers. To facilitate the
formation of the a glycosyl linkage, the amine moiety of galactos-
amine 1 was converted to azide through the copper catalyzed
Department of Clinical Chemistry and Transfusion Medicine,
Sahlgrenska Academy at the University of Gothenburg, Sweden.
E-mail: goran.larson@clinchem.gu.se
c
Department of Psychiatry and Neurochemistry,
Sahlgrenska Academy at the University of Gothenburg, Sweden
Electronic supplementary information (ESI) available: Synthesis procedures,
†
15
diazo transfer reaction (Scheme 1a). Global acetylation followed
by selective removal of the anomeric acetate (Ac) and addition of
characterization data for all new compounds and supplementary figures. See DOI:
0.1039/c4cc05085a
1
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Scheme 2 Synthesis of Ab glycopeptides 9 (3%), 10 (5%) and peptide
1
1 (9%).
2 3 4
Scheme 1 Reagents and conditions: (a) (i) triflic azide, K CO , CuSO ,
H
2
2
O/toluene/MeOH, r.t.; (ii) Ac
steps; (b) hydrazine acetate, DMF, r.t., 82%; (c) trichloroacetonitrile, K
O (1: 1), À30 1C (a only);
O, AcOH, THF, r.t., 56% for 2 steps; (f) H , Pd/C, AcOH, DCM/
MeOH, r.t., 94%. (g) (i) H , Pd/C, p-toluenesulfonic acid monohydrate, THF,
2
O, pyridine, DMAP, 0 1C to r.t., 71% for
2 3
CO ,
Fmoc protected amino acid building blocks including the
glycosylated tyrosines followed by Fmoc deprotection produced
glycopeptides, which were cleaved off the resin using a cocktail
DCM, r.t., 75%; (d) 3, TMSOTf, 4 Å MS, DCM/Et
e) Zn, Ac
2
(
2
2
2
r.t., ii. 2,2,2-trichloroethyl chloroformate, TEA, THF, 0 1C, 80% for 2 steps; of trifluoroacetic acid (TFA), water and triisopropylsilane
h) p-toluenethiol, BF O, DCM, r.t., 85%; (i) NIS, trifluoromethanesulfonic (TIPS). Treatment of the resulting peptides with base removed
ÁEt
acid, 3, 4 Å MS, DCM, À20 1C; (j) Zn, Ac
7: 26%; 4: 26%); (k) H , Pd/C, AcOH, DCM/MeOH, r.t., 90%.
(
3
2
2
O, AcOH, THF, r.t., 52% for 2 steps
all O-acetates on GalNAc and the N-terminal Fmoc. Subsequent
HPLC purification generated pure Ab(1–15) glycopeptides 9 and
(
2
1
0 containing a and b linked GalNAc, respectively. In a similar
trichloroacetonitrile led to the trichloroacetimidate donor 2. manner, the unglycosylated Ab(1–15) 11 was synthesized.
Trimethylsilyl triflate (TMSOTf) promoted glycosylation of Fmoc The synthetic glycopeptides provide valuable standards to
protected tyrosine 3 by donor 2 forming the glycosylated tyrosine, establish MS methods for stereochemical assignment. Ab(1–15)
which was then reduced with simultaneous acetylation leading to glycopeptides 9 and 10 were analyzed by nano-flow liquid
4
as a single stereoisomeric product in 56% yield for the two steps. chromatography electrospray ionization tandem mass spectrometry
3
NMR analysis of 4 ( J
= 3.0 Hz) suggested that the newly (LC-MS/MS or MS2) using a hybrid linear ion trap-Fourier transform
H1–H2
formed glycosidic linkage was a. Catalytic hydrogenolysis of 4 ion cyclotron resonance mass spectrometer. Collision induced
generated tyrosine carboxylic acid 5, which was ready for glyco- dissociation (CID) was utilized to achieve glycosidic and partial
peptide synthesis.
peptide fragmentation and electron capture dissociation (ECD)
In order to form b-linked glycosyl tyrosine, trichloroethyl was utilized for determining the amino acid position of the
carbamate (Troc) protected galactosamine thioglycosyl donor 6 was glycan. The CID-MS/MS (MS2) spectra were collected in the linear
prepared from D-galactosamine hydrochloride 1 (Scheme 1b). Using ion trap (Fig. 1A for glycopeptide 9 and Fig. 1B for glycopeptide 10)
4+
N-iodosuccinimide (NIS) and trifluoromethane sulfonic acid as the resulting from the [M + 4H] precursors (Fig. S1, ESI†). The
4+
promoters, donor 6 was coupled with tyrosine acceptor 3, which theoretical mass (monoisotopic) for the [M + 4H] precursor of
was followed by reduction of the 2-N-trichlorethoxycarbonyl (Troc) compounds 9 and 10 is m/z 508.2213. The measured masses for
group and acetylation leading to two anomeric isomers 7 and 4 in a compound 9 and compound 10 were m/z 508.2207 (1.14 ppm off)
1
7
:1 ratio. 7 and 4 were separated by silica gel chromatography and and 508.2210 (0.63 ppm off), respectively. Both compounds lost
was determined to be the b anomer ( JH1–H2 = 8.5 Hz) by H-NMR GalNAc + H from the [M + 3H] peptide ion (m/z 609.6). Although
3
1
+
3+
spectroscopy. The formation of a large quantity of 4 in the presence qualitatively the spectra were similar, quantitative analysis revealed
of the 2-N-Troc moiety capable of neighboring group participation key differences distinguishing the two structures. For 9, the
4
+
in donor 6 was presumably due to the higher thermodynamic [M + 4H] peptide ion (m/z 457.7), resulting from the neutral
stability of 4 resulting from the anomeric effect. 7 was subjected to loss of GalNAc, had lower intensity than a number of GalNAc
2
+
catalytic hydrogenolysis yielding the b-linked tyrosine carboxylic substituted b- and y-ions (e.g., [y14 + GalNAc] at m/z 479.8)
4
+
acid 8 (Scheme 1c).
(Fig. 1A). The reverse was observed for 10 where the [M + 4H]
2
+
With the glycosyl tyrosines 5 and 8 in hand, glycopeptide ion was more prominent compared to the [y14 + GalNAc] ion
syntheses were performed using the solid phase method on (Fig. 1B). The ratios for the m/z 457.7 and 479.8 ions were found
2-chlorotrityl resin (Scheme 2). Iterative peptide coupling using to be 0.5 Æ 0.1 and 2.6 Æ 0.7 (m Æ SD) for 9 and 10 respectively.
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+
loss of HexNAc + H and a spectrum closely matching that of
CID-MS2 of 9 (Fig. 1C). Specifically, CID-MS3 spectra of the native
2+
glycopeptide possessed a more abundant [ y14 + GalNAc] ion
4+
(
m/z 479.8) and a less abundant [M + 4H] peptide ion (m/z 457.7)
with the intensity ratio of 0.4 Æ 0.2 for the peak at m/z 457.7 over
that at m/z 479.8. Thus, we conclude that the native Ab(1–15)
glycopeptides from CSF are composed of an a-linked HexNAc
moiety, most likely an a-GalNAc moiety. An electron capture dis-
4+
sociation (ECD) MS spectrum of the [M + 4H] ion for compound 9
also confirmed the peptide sequence and pinpointed that the
GalNAc residue was attached to Tyr10 (Fig. S3, ESI†).
17–19
Ab peptides can bind with metal ions,
which can facilitate
nucleation contributing to plaque formation. At the same time, as
+
ions such as free Cu are highly toxic to cells, it has been proposed
2
0
that Ab may play a neuroprotective role by scavenging metal ions.
+
Thus, the Cu binding abilities of Ab may influence plaque biology.
+
This prompted us to analyze the affinity of glycopeptide 9 with Cu
21
3 2
ions through a competitive binding assay. Cu(NO ) was reduced
+
by sodium ascorbate to Cu , which formed an orange complex with
disodium bathocuproinedisulfonic acid (BC) with an absorbance
maximum at 483 nm. When increasing amounts of Ab glycopeptide
9
were added to a solution of the BC–Cu complex, it competed with
+
BC for Cu binding thus serially reducing the absorbance at 483 nm
Fig. 2). Based on absorbance changes, the dissociation constant of
(
+
À14
the [Cu –9] complex was calculated to be 1.69 Æ 0.84 Â 10
M. In
comparison, the dissociation constant of the unglycosylated Ab
+
À15
peptide 11 with Cu was measured to be 2.72 Æ 1.26 Â 10
M,
which was similar to the reported value of Ab (1–16) binding with
+
21
+
20
Cu . The Ab peptide binds Cu in a linear bis-His geometry,
thus most likely the Cu ion is ligated to His13 and His14 in
+
unglycosylated Ab peptide 11. The presence of a bulky GalNAc on
+
Tyr10 in glycopeptide 9 could possibly sterically hinder Cu
+
binding. The impact of reduced Cu affinity of the glycopeptide
Fig. 1 CID-MS2 of Ab(1–15) glycopeptides 9 (A) and 10 (B); and CID-MS3 on plaque formation and toxicity will need to be established
of HexNAc substituted Ab(1–15) from human cerebrospinal fluid (C). The
in vivo in the future.
spectra are intensity zooms of the corresponding CID-MS2 and CID-MS3
In conclusion, we developed viable synthetic routes to the first
spectra (see Fig. S1 and S2, ESI†). Squares symbolize free or glycopeptide-linked
1
15
synthesis of Tyr O-glycosylated Ab peptides. Aided by these well-
HexNAc and horizontal lines represent the peptide DAEFRHDSGYEVHHQ.
defined synthetic samples and tandem MS analysis, we identified
Thus, GalNAc was more easily cleaved from the Tyr residue during
CID when it was attached to the peptide in b-configuration
(glycopeptide 10) compared to the a-configuration (glycopeptide
9). This is most likely due to the higher stability of the glycosidic
linkage in 9 as a result of the anomeric effect. Therefore, MS can be
used to reliably differentiate these two isomeric glycopeptides.
The native Ab(1–15) glycopeptide isolated from human
cerebrospinal fluid (CSF) was examined next by MS. Ab peptides
and glycopeptides were immunopurified using the 6E10 antibody
and any terminal Neu5Ac residues were selectively hydrolyzed
using 0.1 M formic acid at 353 K for 30 minutes resulting in Ab
glycopeptides rich in de-sialylated HexHexNAc (Hex denotes a
1
6
4+
hexose). The [M + 4H] precursor of Ab(1–15) + HexHexNAc
was observed at m/z 548.7350 (Fig. S2A, ESI†) and CID-MS2
resulted in an abundant neutral loss of Hex into Ab(1–15) +
HexNAc at m/z 508.4 (Fig. S2B, ESI†) isomeric to 9 and 10.
+
Fig. 2 Competitive chelation of BC and Ab glycopeptide 9 with Cu .
3
À
Increasing concentrations of glycopeptide 9 disrupted the [CuBC ]
2
Further consecutive fragmentation (CID-MS3) led to a prominent complex resulting in reduction of the absorbance at 483 nm.
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that Tyr10 and O-glycan were most likely linked through an
a-GalNAc linkage in natural Ab glycopeptide fragments isolated from
AD patients. Glycosylation could significantly impact the properties
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