Chemical synthesis of highly congested gp120 V1V2 N -glycopeptide antigens for potential HIV-1-directed vaccines

Article abstract of DOI:10.1021/ja405990z

Critical to the search for an effective HIV-1 vaccine is the development of immunogens capable of inducing broadly neutralizing antibodies (BnAbs). A key first step in this process is to design immunogens that can be recognized by known BnAbs. The monoclonal antibody PG9 is a BnAb that neutralizes diverse strains of HIV-1 by targeting a conserved carbohydrate-protein epitope in the variable 1 and 2 (V1V2) region of the viral envelope. Important for recognition are two closely spaced N-glycans at Asn¹⁶⁰ and Asn¹⁵⁶. Glycopeptides containing this synthetically challenging bis-N-glycosylated motif were prepared by convergent assembly, and were shown to be antigenic for PG9. Synthetic glycopeptides such as these may be useful for the development of HIV-1 vaccines based on the envelope V1V2 BnAb epitope.

Full text of DOI:10.1021/ja405990z

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Article
Chemical synthesis of highly congested gp120 V1V2 N-
glycopeptide antigens for potential HIV-1–directed vaccines
Baptiste Aussedat, Yusuf Vohra, Peter Kinam Park, Alberto Fernandez-Tejada, S. Munir
Alam, S. Moses Dennison, Frederick H. Jaeger, Kara Anasti, Shelley Stewart, Julie H.
Blinn, Hua-Xin Liao, Joseph Gerard Sodroski, Barton F Haynes, and Samuel J. Danishefsky
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja405990z • Publication Date (Web): 05 Aug 2013
Downloaded from http://pubs.acs.org on August 13, 2013
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Chemical synthesis of highly congested gp120 V1V2 N-
glycopeptide antigens for potential HIV-1–directed vac-
cines
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Baptiste Aussedat,a,† Yusuf Vohra,a,† Peter K. Park,a Alberto Fernández-Tejada,a S. Munir Alam,c S. Mo-
ses Dennison,c Frederick H. Jaeger,c Kara Anasti,c Shelley Stewart,c Julie H. Blinn,c Hua-Xin Liao,c,d
Joseph G. Sodroski,f,g,h,i Barton F. Haynes,c,d,e and Samuel J. Danishefsky.a,b*
aLaboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, New
York 10065, United States
bDepartment of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, United States
cDuke Human Vaccine Institute, dDepartment of Medicine, eDepartment of Immunology, Duke University Medical Center,
Durham, North Carolina 27710, United States
fDepartment of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, gDepartment of Microbiology and Immunobi-
ology, Harvard Medical School, Boston, Massachusetts, 02215, United States
hDepartment of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, 02115, Unit-
ed States
iRagon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Cambridge, Mas-
sachusetts, 02139, United States
Supporting Information Placeholder
ABSTRACT: Critical to the search for an effective HIV-1 vaccine is the development of immunogens capable of inducing broadly
neutralizing antibodies (BnAbs). A key first step in this process is to design immunogens that can be recognized by known BnAbs.
The monoclonal antibody PG9 is a BnAb that neutralizes diverse strains of HIV-1 by targeting a conserved carbohydrate-protein
epitope in the variable 1 and 2 (V1V2) region of the viral envelope. Important for recognition are two closely spaced N-glycans at
Asn160 and Asn156. Glycopeptides containing this synthetically challenging bis-N-glycosylated motif were prepared by convergent
assembly, and were shown to be antigenic for PG9. Synthetic glycopeptides such as these may be useful for the development of
HIV-1 vaccines based on the envelope V1V2 BnAb epitope.
quently, a co-crystal structure of PG9 with gp120 variable
regions 1 and 2 (V1V2) grafted onto a mini-protein scaffold
INTRODUCTION
Few important structures in nature are more heavily glyco-
sylated than is the envelope spike (Env) of human immunode-
revealed that the antibody engages high mannose glycans at
Asn160 and Asn156 and an adjacent β-strand (Figure 1A).7 In
ficiency virus type 1 (HIV-1).1 A multitude of designed con-
contrast to 2G12, which apparently does not interact with the
structs that might simulate the unique architecture of Env have
been considered and pursued in the context of potential HIV-
gp120 peptide backbone, PG9 binds an epitope that contains
both carbohydrate and peptide components, while possessing
1–directed vaccines. Yet, until recently, the only template for
a normal heavy chain arrangement.
immunological recognition of this dense “glycan shield” has
been the broadly neutralizing antibody (BnAb) 2G12.2 Follow-
In light of our continuing involvement in the synthesis of
glycoproteins and complex glycopolypeptide motifs,8 we re-
garded these new structural observations with particular inter-
est. It seemed not unlikely that successful design of vaccines
based on the PG9 epitope would depend crucially on close
simulation of the detailed surface glycopeptide architecture of
Env. Given the importance of the glycan domains in forming
this conserved epitope, it seemed that access to Env constructs
that are well-defined and homogeneous with respect to glyco-
sylation state would greatly facilitate immunogen development
efforts. Past work has largely relied on recombinant Env prep-
arations, supplied as mixtures of glycoforms.9 This serious
heterogeneity complicates efforts to draw precise correlations
between glycan composition and immunoactivity. Absent a
detailed understanding of the structural biology of the prob-
lem, informed vaccine design is, naturally, much more diffi-
cult. We were stirred by the prospect that de novo chemical
synthesis could provide the complex, yet homogeneous probe
substrates needed for rationally based advances in this urgent
endeavor.
ing its discovery, many laboratories,3 including our own,4 were
able to generate mimics of the oligomannose cluster that con-
stitutes its epitope, in the hope of eliciting 2G12-like antibod-
ies. Unfortunately, these efforts were not successful. While
many factors have been cited to explain the general difficulties
surrounding BnAb induction,5 the case of 2G12 is likely com-
plicated further by the unusual domain-exchanged arrange-
ment of its heavy chains, which is thought to be responsible
for its unique mode of glycan recognition.2d
In 2009, two new and potent BnAbs, PG9 and PG16, were
isolated from an HIV-1–infected donor from sub-Saharan Af-
rica.6 These monoclonal antibodies (mAbs) were found to
neutralize 70–80% of circulating HIV-1 isolates. Initial
epitope mapping suggested that PG9 and PG16 were targeting
a new glycan-dependent Env epitope, entirely distinct from
that of 2G12. A sensitivity to quarternary structure was also
noted, as these BnAbs exhibited a preference for binding fully
assembled trimeric viral spike over monomeric Env. Subse-
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More specifically, we hypothesized that fully synthetic, ho-
A!
B!
N156!
glycan !
mogeneous gp120 V1V2 polypeptide domains, bearing de-
fined glycosyl patterns, might be able to function as minimal
mimics of the PG9 epitope. If such uniform, synthetically-
derived constructs were able to simulate the conformation of
the pertinent native envelope glycoproteins, they would pro-
vide a logical starting point for immunogen design. Moreover,
a minimal construct could, in theory, present the desired BnAb
epitope without interference from other potentially more im-
munogenic Env determinants.5
1
2
Greek key motif!
C!
V2!
loop!
3
4
5
6
B!
A!
D!
C!
B!
V1!
loop!
V1 loop!
A!
D!
7
N160!
glycan !
8
9
Herein, we describe the chemical synthesis of gp120 V1V2
glycopeptides as single glycoforms10 that were found to bind
the BnAb PG9 with surprisingly high affinities. During the
course of this work, we had to deal with and overcome the
fundamental synthetic challenge arising from the close spacing
of large glycans along the peptide backbone. In engaging this
challenge, we would be pressing against the limits of the prior
art we had developed in the realm of glycopeptide ligations.11
V2 loop!
C!
10
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Man
OH
O
HO
HO
HO
GlcNAc
OH
O
O
HO
O
HO
HO
O
O
OH
O
O
OH
HO
HO
HO
O
HO
O
O
HO
O
H
N
O
HO
NHAc
HO
HO
!
NHAc
O
OH
HO
N
H
C
O
Asn
RESULTS AND DISCUSSION
Design and Strategy. The structure of the gp120 V1V2
domain in the context of a bound PG9 mAb Fab consisted of
four anti-parallel β-strands (A–D) that folded into what is
known as a Greek key motif (Figure 1B).7 Based on these x-
ray crystallographic data, PG9 makes contacts with the C β-
strand, and with the Man5GlcNAc2 glycans12 (Figure 1C) at
Asn160 and Asn156, which reside on strand B. Since most of the
structural features recognized by PG9 appear to be localized
on the B and C strands,13 we reasoned that an epitope mimic
should, at the very least, encompass this region. Our initial
prototype is shown in Figure 1D. The 35-amino acid peptide
corresponds to positions 148–184 of gp120 (HXB2 number-
ing) derived from the A244 sequence,14 an Env variant that is
known to bind PG9 in monomeric form (i.e., without requiring
trimerization).15 With regard to the glycan structure,
Man5GlcNAc2 was thought to be the best candidate on the
basis of prior studies involving perturbations of glycan pro-
cessing.16,17 The primary target that emerged from this analysis
was glycopeptide 1 with Man5GlcNAc2 units installed at the
two glycosylation sites, Asn160 and Asn156; we also planned to
D!
V1V2 Man5GlcNAc2
148
184
AcHN
ITDEVRNCSFNMTTELRDKKQKVHALFYKLDIVPI
NH2
156
160
1
V1V2 Man3GlcNAc2
148
184
AcHN
AcHN
ITDEVRNCSFNMTTELRDKKQKVHALFYKLDIVPI
NH2
156
160
2
V1V2 GlcNAc2
148
184
ITDEVRNCSFNMTTELRDKKQKVHALFYKLDIVPI
156
NH2
160
3
B!
C!
Figure 1. Design of gp120 V1V2 domain broadly neutralizing
epitope mimics. (A) Crystal structure of a scaffolded V1V2 do-
main from the CAP45 strain of HIV-1 (red ribbons) in complex
with PG9 Fab (gray surface) (PDB ID 3U4E with scaffold hid-
den). The glycans at N160 and N156 are depicted with colored
spheres representing atoms of the mannose (green) and N-
acetylglucosamine (blue) residues. Disulfide bonds are shown as
yellow sticks. Dashed arrows indicate where the disordered region
of the V2 loop would be connected. Figure was created using
PyMOL. (B) Schematic of the Greek key topology of the V1V2
domain. Strands are represented as arrows and disulfide bonds as
yellow bars. (C) Chemical structure of Man5GlcNAc2–Asn. (D)
Structures of candidate BnAb antigens, derived from residues
148–184 of the A244 strain gp120 (HXB2 numbering), encom-
passing the B and C β-strands (approximate location shown with
red arrows) of the V1V2 domain, and bearing two N-linked
Man5GlcNAc2, Man3GlcNAc2, or GlcNAc2 oligosaccharides.
gain access to simpler glycoforms
2 and 3 bearing
Man3GlcNAc2 and chitobiose (GlcNAc2), respectively.18 These
could be used to probe the importance of the outer mannose
residues for recognition.
We term the general synthetic approach that our laboratory
has applied to complex glycoprotein targets as convergent
assembly.11 In our usual modus operandi, N-linked sugars are
installed via aspartylation of unprotected glycosyl amines,
drawing from the precedent of Lansbury,19 which we20 and
others21 have extended in substantive ways. As we examined
goal structures 1–3 in particular, we noted that the close spac-
ing of the two glycans, especially with larger oligosaccharides,
could present a difficult challenge for their incorporation. We
anticipated that application of our methods in this demanding
context would afford valuable teachings regarding the synthe-
sis of the required clustered N-glycan motifs. As for the sugars
themselves, Man3GlcNAc2 constitutes the common pentasac-
charide core of all N-glycans; it has been synthesized previ-
ously by our laboratory and others.22 By contrast,
Man5GlcNAc2 seems to have received less attention as a syn-
thetic target.23,24 We start by describing our route to the desired
glycans.
Synthesis of Man5GlcNAc2 and Man3GlcNAc2 glycans.
Our studies commenced with experiments directed to the syn-
thesis of the Man5GlcNAc2 heptasaccharide. The β-mannosyl
linkage of the core trisaccharide 6 was constructed as we have
in prior contexts25 by uniting Crich donor 426,27 with chitobiose
acceptor 525b (Scheme 1). Fortunately, the minor quantities of
undesired α-isomer formed (<10%) could be separated by
careful chromatography to afford trisaccharide 6 as a single
diastereomer in 86% yield. The PMB group was removed in
83% yield. Coupling of the resulting acceptor 7 with thiogly-
coside donor 8 was accomplished under NIS/TMSOTf activa-
tion conditions, yielding tetrasaccharide 9. Cleavage of the
benzylidene acetal with aqueous acetic acid afforded diol 10 in
63% overall yield from 7.
Scheme 1. Synthesis of tetrasaccharide 10.a
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OdfBz
BnO
BnO
HO
BnO
OBn
O
Ph
O
O
a
BnO
O
O
BnO
BnO
HO
HO
O
PMBO
O
BnO
OBn
O
+
OBn
1
2
3
BnO
NPhth
O
BnO
OdfBz
O
NPhth
4
O
O
BnO
O
O
S(O)Ph
5
O
BnO
+
BnO
O
OBn
STol
O
NPhth
BnO
BnO
10
NPhth
O
OdfBz
BnO
BnO
SEt
BnO
BnO
BnO
15
O
OdfBz
OBn
O
8
Ph
O
BnO
BnO
OdfBz
BnO
4
O
O
BnO
O
BnO
RO
O
O
BnO
OBn
5
6
7
8
NPhth
OdfBz
O
c
BnO
BnO
a
NPhth
6
7
R = PMB
R = H
b
BnO
OdfBz
O
O
BnO
O
OBn
O
RO
RO
O
BnO
O
BnO
O
BnO
BnO
BnO
BnO
O
O
O
BnO
OBn
O
O
OdfBz
OBn
9
BnO
HO
NPhth
O
BnO
BnO
O
BnO
O
NPhth
O
BnO
O
BnO
O
OdfBz
10
11
12
13
OBn
BnO
9
R = CHPh
NPhth
BnO
BnO
d
NPhth
O
OdfBz
10 R = H
BnO
16
aReagents and conditions: (a) Tf2O, DTBP, MS AW-300,
CH2Cl2, –78 °C, 86%; (b) DDQ, CH2Cl2, H2O, 83%; (c) thiogly-
coside 8, NIS/TMSOTf, MS AW-300, CH2Cl2, 0 °C→r.t.; (d)
OH
O
HO
HO
HO
b–e
14
15
16
17
18
19
20
21
22
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OH
O
O
HO
O
AcOH, H2O, 63% (2 steps).
HO
HO
O
OH
O
O
OH
HO
HO
HO
O
Assembly of the heptasaccharide at first in protected form
was accomplished convergently by selective mannosylation at
C-6 of 10 with branched donor 15. Synthesis of the requisite
trisaccharide 15 was achieved by elaboration of mannosyl
building block 11 (Scheme 2). Reductive ring opening was
accomplished selectively with borane-THF complex in the
presence of copper triflate in 96% yield.28 Cleavage of the
PMB group afforded the 3,6-diol, 13, which underwent double
mannosylation with imidate donor 14 to furnish the bis-α-
mannosylated trisaccharide 15 in 75% yield.
O
HO
O
HO
O
O
HO
XH
NHAc
HO
HO
NHAc
O
OH
HO
17
18
X = O
f
X = NH
aReagents and conditions: (a) NIS/TMSOTf, MS AW-300,
CH2Cl2, 0 °C→r.t., 64%; (b) NaOMe, MeOH, CH2Cl2; (c)
H2NCH2CH2NH2, n-BuOH, PhMe, 90 °C; (d) Ac2O, Et3N,
MeOH; (e) H2, Pd(OH)2/C, MeOH, H2O, 77% (4 steps); (f) sat.
aq. NH4HCO3, 40 °C, quantitative.
Scheme 2. Synthesis of trisaccharide donor 15.a
The pentasaccharide, Man3GlcNAc2, was obtained from
tetrasaccharide intermediate 10 by selectively coupling donor
8 to the C-6 hydroxyl group (Scheme 4). Although this reac-
tion was complicated by a small amount of bis-glycosylation,
the protected Man3GlcNAc2 unit was isolated in 94% yield.
Subjection of this material to the 4-step global deprotection
protocol described above resulted in a 74% overall yield of
fully deprotected pentasaccharide 19 as a mixture of anomers.
The β-anomeric amine 20 was subsequently generated by ap-
plication of the Kochetkov conditions.
OdfBz
O
OdfBz
O
OdfBz
O
Ph
O
HO
BnO
PMBO
HO
BnO
HO
a
b
O
PMBO
11
12
13
SEt
SEt
SEt
OdfBz
O
BnO
BnO
BnO
OdfBz
O
OdfBz
O
HO
BnO
HO
BnO
BnO
BnO
c
OdfBz
O
O
+
BnO
O
O
13
14
SEt
O
NH
BnO
BnO
SEt
15
CCl3
BnO
OdfBz
Scheme 4. Synthesis of pentasaccharide 20.a
aReagents and conditions: (a) Cu(OTf)2, BH3·THF, THF, 0 °C,
96%; (b) DDQ, CH2Cl2, H2O, 89%; (c) TMSOTf, MS AW-300,
CH2Cl2, 0 °C, 75%.
OH
HO
O
HO
HO
With the stage set for the key coupling, 15 was activated
(NIS/TMSOTf) and joined with 10, thus providing the fully
elaborated protected heptasaccharide 16 in 64% yield (Scheme
3). A four-step sequence involving ester saponification,
phthalimide cleavage, N-acetylation, and hydrogenolysis pro-
ceeded smoothly to give fully deprotected heptasaccharide 17
as a mixture of anomeric alcohols in 77% yield. This com-
pound underwent apparently quantitative conversion to the β-
anomeric amine 18 under Kochetkov amination conditions.29
O
a–e
OH
O
+
8
10
HO
HO
O
HO
O
O
O
O
HO
XH
HO
NHAc
HO
HO
NHAc
O
OH
HO
19
20
X = O
X = NH
f
aReagents and conditions: (a) NIS/TMSOTf, MS AW-300,
CH2Cl2, 0 °C→r.t., 94%; (b) NaOMe, MeOH, CH2Cl2; (c)
H2NCH2CH2NH2, n-BuOH, PhMe, 90 °C; (d) Ac2O, Et3N, MeOH,
95%; (e) H2, Pd(OH)2/C, MeOH, H2O, 74% (4 steps); (f) sat. aq.
NH4HCO3, 40 °C, quantitative.
Scheme 3. Synthesis of heptasaccharide 18.a
Convergent assembly of V1V2 glycopeptides. The most
risky phase of the effort involved the assembly of the peptide
domain of the targeted glycopeptide constructs, and their cou-
pling to different oligosaccharides. Two basic strategies were
considered. Our first thoughts envisioned installing both gly-
cans simultaneously on the full-length peptide, bearing in
mind our prior successes with two- and three-fold aspartyla-
tions on cyclic scaffolds.4d Pilot experiments using chitobiose
as a model glycan, however, yielded only unmanageable mix-
tures of mono- and bis-glycosylated forms, presumably due to
the steric demands imposed by the close proximity of aspar-
tylation sites. Anticipating that driving the reaction to comple-
tion with larger oligosaccharides might require a substantial
excess of precious glycosyl amine, we decided to pursue an
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alternative approach involving the ligation of two pre-built
mation of succinimide (via cyclization of the asparagine side
glycopeptide fragments. Here, the presence of Cys157 served to
raise the possibility of native chemical ligation (NCL).30
chain nitrogen onto the thioester). While certainly less than
optimal, these ligations represent, to the best of our
knowledge, the first examples of NCL with peptide thioesters
carrying an N-glycan directly at the C-terminus. Furthermore,
no other syntheses have been reported of linear glycopeptides
bearing such closely spaced N-glycans, i.e., separated by three
amino acids or less.34 While the yields for the overall sequence
are likely to benefit from optimization,35 our concerns at first
were focused more on the purity of the synthetic constructs
rather than on maximizing material throughput. Fortunately,
the synthesis, even in its present form, has produced sufficient
quantities to initiate the biological studies now underway both
in vitro and in vivo to chart a path forward to a clinically eval-
uable HIV-1 vaccine (vide infra).
1
2
3
4
5
6
7
8
We anticipated that this approach would not be without its
own complications, given the close positioning of the glycans.
Indeed, one of the coupling partners (Ile148–Asn156) must carry
the sterically demanding oligosaccharide on its C-terminal
thioester-bearing amino acid. Nevertheless, this scheme was
successfully reduced to practice, as described below.
In the event, N-terminal fragment, peptide thioester 21, was
obtained by Fmoc solid phase peptide synthesis (SPPS) and
post-resin C-terminal functionalization procedures31 used by
our laboratory in the context of other glycopeptide endeavors
(Scheme 5).32 Using our recently reported one-flask aspartyla-
tion/deprotection protocol,20 the free carboxylic acid side chain
at position 156 was joined to the Man5GlcNAc2 glycosyl
amine 18, followed by TFA treatment to provide glycopeptide
thioester 22 in 44% yield after purification by reversed-phase
HPLC. The formation of a side product of identical mass was
observed in small quantities (5–10%), presumably due to base-
induced epimerization of the thioester during the aspartylation.
Fortunately, it could be easily separated during the purifica-
tion.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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Antigenicity studies. To assess the extent to which our syn-
thetic V1V2 glycopeptides are able to recapitulate the mAb
PG9 V1V2 BnAb epitope, we studied the binding of con-
structs 1–3 to PG9 by surface plasmon resonance (SPR) analy-
sis (Figure 2). PG9 was captured by surface-immobilized anti-
human Ig Fc, and the V1V2 glycopeptide constructs were in-
jected as analytes on BIAcore 3000 instruments as described
previously.36 We found that the Man5GlcNAc2 V1V2 (1) and
Man3GlcNAc2 V1V2 (2) glycopeptides both exhibited signifi-
cant affinity for mAb PG9 (Figures 2A and 2B), with Kd’s of
311 and 119 nM, respectively (obtained by using a global fit
of multiple titrations to a 1:1 Langmuir model). By contrast,
the chitobiose-bearing construct 3 did not bind mAb PG9
(Figure 2C), suggesting that the presence of α-linked mannose
residues on the glycans is important for recognition. Further-
more, binding by the unglycosylated V1V2 peptide (i.e.,
“aglycone”) (Figure 2D) or the solitary protein-free
Man5GlcNAc2 and Man3GlcNAc2 oligosaccharides was not
detected (Figure 2E and F). Mixtures of “aglycone” and gly-
can similarly failed to show measurable binding (not shown).
For the C-terminal fragment, a similar one-flask sequence
was used to convert protected peptide 23 to deprotected gly-
copeptide 24 in 22% yield. As has been previously observed,
emplacement of a pseudoproline motif at Thr162 (n + 2 relative
to Asp160) was helpful in suppressing undesired aspartimide
formation during the aspartylation.20,21 The isolated yield for
this fragment was eroded by factors that complicated the final
purification of glycopeptide 24, including near overlap of the
unglycosylated peptide, and the persistence of capped trunca-
tion products that had formed during the course of the SPPS
(by an as yet undefined mechanism). Despite these obstacles,
sufficient quantities of fragments of 22 and 24 could be syn-
thesized and joined by NCL to afford the fully elaborated gly-
copeptide 1 bearing Man5GlcNAc2 units at Asn160 and Asn156 in
55% yield. The simpler glycoforms 2 and 3, possessing two
Man3GlcNAc2 and two chitobiose glycans, respectively, were
prepared by an analogous route (see Supporting Information
for details).
A!
C!
E!
B!
V1V2 Man5GlcNAc2!
Kd = 311 nM!
V1V2 Man3GlcNAc2!
Kd = 119 nM!
250
200
150
100
50
200
150
100
50
0
0
-50
-50
-50 0 50100 200 300 400 500 600 700 800
-50 0 50100 200 300 400 500 600 700 800
Scheme 5. Synthesis of V1V2 glycopeptide 1.a
D!200
200
V1V2 GlcNAc2!
V1V2 aglycone!
150
100
50
150
100
50
TrtS
O
OH
156
O
OH
tBu
Mpe
Pbf Boc Dmcp Trt
Boc
148
157
184
AcHN
ITDEVRD
SEt
BocHN
NH2
CSFDMTTELRDKKQKVHALFYKLDIVPI
tBu Pbf
tBu
tBu
tBu Boc Boc
tBu
tBu
!
!
21
23
aspartimide formation
prevented by pseudoproline
at consensus Ser/Thr site
0
0
One-flask
aspartylation/
deprotection
-50
0
100
200
300
400
500
600
-50
0
100
200
300
400
500
600
a,b
c,d
F!
Man3GlcNAc2 glycan!
Man5GlcNAc2 glycan!
50
40
30
20
10
0
50
40
30
20
10
0
HS
148
156
157
184
AcHN
ITDEVRN
SEt
H2
N
CSFNMTTELRDKKQKVHALFYKLDIVPI
NH2
22
24
Native chemical
ligation
e
-50
0
100
200
300
400
500
600
-50
0
100
200
300
400
500
600
Time (s)!
Time (s)!
148
184
Figure 2. Binding of mAb PG9 to gp120 V1V2 glycopeptides.
SPR sensorgrams showing binding of mAb PG9 to V1V2 glyco-
peptides derivatized with Man5GlcNAc2 (A) and Man3GlcNAc2
(B). V1V2 Man5GlcNAc2 binding curves are shown for glycopep-
tide concentrations at 5, 10, 20, 30 and 40 μg/mL and V1V2
Man3GlcNAc2 at 1, 2, 5, 10 and 20 μg/mL. Control SPR sen-
sograms showing minimal to no binding of mAb PG9 to V1V2
GlcNAc2 (C), V1V2 aglycone (D), Man5GlcNAc2 glycan alone
(E), and Man3GlcNAc2 glycan alone (F). V1V2 GlcNAc2 and
aglycone peptides were injected at 200 μg/mL (C, D) and
Man5GlcNAc2 and Man3GlcNAc2 glycans at 25 μg/mL (E, F) over
PG9 captured on anti-human IgG (Fc-specific) surfaces. SPR data
were derived following subtraction of non-specific signal on a
control anti-RSV mAb (Synagis, red curve in C–F).
AcHN
ITDEVRNCSFNMTTELRDKKQKVHALFYKLDIVPI
NH2
1
aReagents and conditions: (a) Man5GlcNAc2–NH2 (18),
PyAOP, DIEA, DMSO; (b) Cocktail 88:5:5:2
TFA/phenol/water/triisopropylsilane, 44% (2 steps); (c)
Man5GlcNAc2–NH2 (18), PyAOP, DIEA, DMSO; (d) Cocktail R
= 90:5:3:2 TFA/thioanisole/ethanedithiol/anisole, 22% (2 steps);
(e) 6 M Gnd·HCl, 200 mM Na2HPO4, 200 mM MPAA, 20 mM
TCEP·HCl, pH 7.2, 55%.
B
=
In all cases, the final ligation proved to be difficult. Indeed,
three equivalents of thioester were required for the reaction to
progress to completion.33 Careful control of the reaction pH
was needed to avoid apparent epimerization or excessive for-
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Taken together, these data demonstrate that PG9 recognition
of our V1V2 constructs is critically dependent on both the
ASSOCIATED CONTENT
1
2
3
4
5
6
7
8
Supporting Information. Detailed experimental procedures,
including spectroscopic and analytical data for new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
peptide and carbohydrate domains. Covalent linkage between
them is essential, since the apparent affinities for each individ-
ual component in isolation are very low. Indeed, NMR studies
have shown that the Kd for binding of PG9 to Man5GlcNAc2–
Asn alone is ~1–2 mM,6 consistent with the general trend that
individual protein-carbohydrate interactions tend to be weak.
The overall high “avidity” observed may be attributed to the
synergies afforded by multivalency, wherein the binding to
PG9 is enhanced by multiple simultaneous interactions with
the C β-strand, and the Asn160 and Asn156 glycans.7 Conforma-
tional effects may also play a role, as glycosylation of the pep-
tide backbone could have a favorable orienting influence on
the involved peptide and/or sugar residues.37 Evidence of such
“cross-talk” between peptido and glyco domains has been
observed by our laboratory in other settings.38
AUTHOR INFORMATION
Corresponding Author
*s-danishefsky@ski.mskcc.org
9
Author Contributions
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†B.A. and Y.V. contributed equally.
Notes
The authors declare no competing financial interest.
In light of these findings, it seems likely that proper evalua-
tion of the optimal glycans and peptide sequences for mimick-
ing the V1V2 BnAb epitope—and other similar glycopeptide
antigens—will require their presentation in their native N-
linked context (or as some close isostere). Adopting this ap-
proach enabled us to make the unexpected discovery that the
Man3GlcNAc2-based construct 2 binds PG9 just as well, and
perhaps even a little better, than construct 1 (bearing
Man5GlcNAc2).39 Studies are underway to better understand
the robust recognition of this non-canonical40 glycan by PG9.
In the meantime, we note that such fine structure preferences
were not detected by previous approaches, such as glycan ar-
ray analysis, that interrogated PG9 binding to isolated carbo-
hydrates in the absence of a peptide backbone.41
ACKNOWLEDGMENT
This work was supported by NIH grants for the Center for
HIV/AIDS Vaccine Immunology, AI 067854 and the Duke Center
for HIV/AIDS Vaccine Immunology-Immunogen Discovery, AI
100645. P.K.P. was supported by a postdoctoral fellowship (PF-
11-014-01-CDD) from the American Cancer Society. A.F.-T.
thanks the European Commission (Marie Curie International Out-
going Fellowship) for financial support. The authors thank Dr.
George Sukenick, Ms. Hui Fang, and Ms. Sylvi Rusli of the NMR
Analytical Core Facility at MSKCC for MS and NMR assistance.
We also thank Dr. Ping Wang, Dr. Xiangyang Wu, Dr. Maciej
Walczak, Dr. Gardner Creech, Dr. Steven Townsend, and Ms.
Rebecca Wilson for advice and helpful discussions. We are grate-
ful to Dr. Wayne Koff (International AIDS Vaccine Initiative,
New York, NY) and Dr. Dennis Burton (The Scripps Research
Institute, La Jolla, CA) for provision of mAb PG9.
More profound, perhaps, is the overall question of how the
modestly sized glycopeptides 1 and 2 are able to simulate the
antigenicity of native envelope glycoproteins so well. PG9 and
other BnAbs that target the same V1V2 epitope (e.g., PG16
and CH01 to CH0415) are thought to be sensitive to quarter-
nary structure, binding Env trimers better than monomeric
Env.6,13 Indeed, relatively few Env sequences are known to
bind PG9 in monomeric form, so it is noteworthy that compar-
atively small (6–7 kDa), linear 35-mer Env glycopeptide
fragments like 1 and 2 are able to bind PG9 with respectable
affinities, with Kd’s on the order of 10-7 M.42 Such affinities
compare favorably with the published Kd’s of ~5 × 10-8 M for
the full-length A244 Env monomer (from which 1 and 2 are
derived),15 and ~10-8 M for a recently reported trimeric Env
construct.13 We are actively investigating the nature of the
binding interaction with PG9; preliminary results suggest that
it cannot be fully explained by a simple “induced-fit” mode of
association.
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CONCLUSION
In summary, we have designed and chemically synthesized
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BnAb PG9. Key to these initial successes were significant
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immunogenicity in animal models. Similar and perhaps even
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BnAb epitopes.43 This is an ongoing program whose results
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ed ethyl thioester can be recovered after the reaction.
(34) Closely spaced N-linked glycans on cyclic peptide scaffolds
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Pancera, M.; Shahzad-ul-Hussan, S.; Doria-Rose, N. A.; McLellan, J.
(35) Work is also in progress to evaluate alternative synthetic strat-
egies that would yield a more “process-friendly” route.
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(37) The conformational consequences of N-glycosylation have
been well studied. For reviews, see: (a) Imperiali, B.; O’Connor, S. E.
Curr. Opin. Chem. Biol. 1999, 3, 643–649. (b) Meyer, B.; Möller, H.
Conformation of Glycopeptides and Glycoproteins. In Glycopeptides
and Glycoproteins: Synthesis, Structure, and Application; Wittmann,
V., Ed.; Topics in Current Chemistry, Vol. 267; Springer: Berlin,
2007; pp 187–251.
(38) (a) Live, D. H.; Kumar, R. A.; Beebe, X.; Danishefsky, S. J.
Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 12759–12761. (b) Live, D.
H.; Wang, Z.-G.; Iserloh, U.; Danishefsky, S. J. Org. Lett. 2001, 3,
851–854.
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(39) Similarly, using V1V2 glycopeptide isoforms bearing natively
N-linked sugars, Amin et al. (ref. 10b) detected an astonishing prefer-
ence of PG9 for a sialylated complex-type glycan at Asn156
.
(40) Man3GlcNAc2, also known as “paucimannose,” is more char-
acteristically associated with plant and invertebrate glycoproteins,
although some have argued that it can be expressed in certain patho-
logical states in mammals, such as inflammation or malignancy: Zips-
er, B.; Bello-DeOcampo, D.; Diestel, S.; Tai, M.-H.; Schmitz, B. J.
Carbohydr. Chem. 2012, 31, 504–518.
(41) Others have remarked on the limitations of glycan array anal-
ysis in delineating BnAb anti-glycan specificities: “Protein-free gly-
can binding by anti-HIV antibodies is not always detectable; e.g.,
although PG9 recognizes a gp120-associated high-mannose glycan,
no binding to protein-free glycans was detected in microarrays. Thus,
although a positive result in a glycan microarray implies involvement
of a particular glycan in an antibody epitope, a negative result does
not rule out glycan recognition.” For the full discussion, see: Mou-
quet, H.; Scharf, L.; Euler, Z.; Liu, Y.; Eden, C.; Scheid, J. F.; Halper-
Stromberg, A.; Gnanapragasam, P. N. P.; Spencer, D. I. R.; Seaman,
M. S.; Schuitemaker, H.; Feizi, T.; Nussenzweig, M. C.; Bjorkman, P.
J. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, E3268–E3277.
(42) As a point of comparison, we note that Amin et al. (ref. 10b)
prepared more than 25 V1V2 glycopeptides bearing various combina-
tions of high mannose and complex-type glycans, and yet their high-
est affinity constructs bound PG9 Fab with Kd’s of ~5 × 10-6 M.
(43) Walker, L. M.; Huber, M.; Doores, K. J.; Falkowska, E.; Pe-
jchal, R.; Julien, J.-P.; Wang, S.-K.; Ramos, A.; Chan-Hui, P.-Y.;
Moyle, M.; Mitcham, J. L.; Hammond, P. W.; Olsen, O. A.; Phung,
P.; Fling, S.; Wong, C.-H.; Phogat, S.; Wrin, T.; Simek, M. D.; Prin-
cipal Investigators, P. G.; Koff, W. C.; Wilson, I. A.; Burton, D. R.;
Poignard, P. Nature 2011, 477, 466–470.
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N160!
N156!
glycan!
glycan!
Cys!
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De novo
chemical
synthesis!
Asn!
OH
O
HO
HO
HO
OH
Phe!
Val!
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HO
O
HO
HO
Ser!
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OH
NHAc
Lys!
Designed gp120 V1V2
domain epitope mimic!
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