Addition of Sialic Acid to Insulin Confers Superior Physical Properties and Bioequivalence

Article abstract of DOI:10.1021/acs.jmedchem.0c00266

Native insulin is susceptible to biophysical aggregation and fibril formation, promoted by manual agitation and elevated temperatures. The safety of the drug and its application to alternative forms of administration could be enhanced through the identification of chemical modifications that strengthen its physical stability without compromising its biological properties. Complex polysialic acids (PSAs) exist naturally and provide a means to enhance the physical properties of peptide therapeutics. A set of insulin analogues site-specifically derivatized with sialic acid were prepared in an overall yield of 50-60%. Addition of a single or multiple sialic acids conferred remarkable enhancement to the biophysical stability of human insulin while maintaining its potency. The time to the onset of fibrillation was extended by more than 10-fold relative to that of the native hormone. These results demonstrate that simplified sialic acid conjugates represent a viable alternative to complex natural PSAs in increasing the stability of therapeutic peptides.

Full text of DOI:10.1021/acs.jmedchem.0c00266

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Article
Addition of Sialic Acid to Insulin Confers
Superior Physical Properties and Bioequivalence
Daniel Kabotso, David Smiley, John Philip Mayer, Vasily M. Gelfanov,
Diego Perez-Tilve, Richard D. DiMarchi, Nicola L. B. Pohl, and Fa Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00266 • Publication Date (Web): 14 May 2020
Downloaded from pubs.acs.org on May 14, 2020
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Addition of Sialic Acid to Insulin Confers Superior Physical Properties and Bioequivalence
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Daniel E. K. Kabotso,[a,b] David Smiley,[b] John Mayer,[b] Vasily M. Gelfanov,[c] , Diego Perez-Tilve,[d]
Richard D. DiMarchi,[b] Nicola L. B. Pohl*[b], Fa Liu*[e]
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[a]
Dr. D. E. K. Kabotso,
School of Basic and Biomedical Sciences, University of Health and Allied Sciences, PMB 31, Ho, Volta
Region-Ghana
[b]
Prof. N. L. B. Pohl, Mr. D, Smiley, Dr. J. Mayer, Prof. R. DiMarchi
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
email: npohl@indiana.edu
[c]
Dr. V. M. Gelfanov
Novo Nordisk Indianapolis Research Center, 5225 Exploration Dr., Indianapolis, IN 46241
[d]
Dr. D. Perez-Tilve
Department of Pharmacology and Systems Physiology, University of Cincinnati-College of Medicine,
Cincinnati, OH, 45267 USA
[e]
Dr. F. Liu
Novo Nordisk Research Center, 530 Fairview Avenue North, Seattle, Washington 98109, USA
email: falx@novonordisk.com
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Abstract
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Native insulin is susceptible to biophysical aggregation and fibril formation, promoted by manual agitation
and elevated temperatures. The safety of the drug and its application to alternative forms of administration
could be enhanced through identification of chemical modifications that strengthen its physical stability
without compromising its biological properties. Complex polysialic acids (PSAs) exist naturally and
provide a means to enhance the physical properties of peptide therapeutics. A set of insulin analogues site-
specifically derivatized with sialic acid were prepared in overall yield of 50-60%. Addition of a single, or
multiple sialic acids conferred remarkable enhancement to biophysical stability of human insulin, while
maintaining its potency. The time to the onset of fibrillation was extended by more than tenfold relative to
native hormone. These results demonstrate that simplified sialic acid conjugates represent a viable
alternative to complex natural PSAs in increasing the stability of therapeutic peptides.
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Introduction
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Diabetes is a metabolic disease associated with insufficient insulin action that has grown to global
epidemic size.1, 2 The discovery of insulin in 1921 by Banting and Best miraculously turned an untreatable
disease to one that could be managed by daily injection of the hormone.3-5 However, the therapeutic benefit
of insulin has not yet been fully realized for multiple reasons, most notably its narrow therapeutic index
and fragile physical properties. The pursuit of an insulin formulation or analog with enhanced stability
under conditions of elevated temperature and agitation is a medicinal priority. Such an analog would extend
the commercial life of the drug, simplify its transport and storage, and broaden its use most notably in pump
administration.6,7 These attributes would further strengthen the safety of insulin therapy and importantly
extend its benefits in tropical climates or where refrigeration is limited.8
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Several molecular approaches have been reported to enhance the stability of insulin, and these
include single-chain insulin analogs,7 four-disulfide bond insulin analogs,9-10 and polyethylene glycol (PEG)
conjugates.11 The single-chain analogs employ a short peptide to link the A-chain N-terminus to the B-chain
C-terminus. The resulting modification restricts conformational flexibility to minimize the formation of the
fibrillation nucleus, an event that involves a large conformational change from native structure.12 Four-
disulfide bond insulin analogs achieve superior biophysical stability via a mechanism similar to the single
chain analogs. PEGylation utilizes the shielding effect of the hydrophilic polymer, where the large
hydrodynamic radius minimizes the self-association of insulin to inhibit aggregation.13 Although all
approaches can be effective, the potential of the former two is complicated by the risk of immune
recognition of the non-native link and cross-reactivity with other endogenous single-chain insulin related
peptides.7, 9-10 The latter approach is compromised by the slow metabolism of the PEG-polymer leading to
tissue accumulation and associated toxicity.14
Polysialic acid (PSA), facilitates bacterial escape immune surveillance, and has been proposed by
Gregoriadis et al. in 1993 to serve a unique role in encapsulating therapeutic proteins.15 It was proposed
that the hydrophilic nature of PSA forms an aqueous layer that serves to prevent the interaction of the
protein backbone with the biological milieu to enhance biological activity.15-17 Given its natural character,
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PSA has the additional virtue in being biodegradable minimizing the risk that otherwise plaques synthetic
polymers with poor clearance from ligand directed target tissues. As such, PSA has been chemically
conjugated to a number of proteins, including asparaginase, interferon alpha-2b, erythropoietin and insulin
to increase stability, extend duration of action, and enhance therapeutic efficacy.16, 18-21
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The use of naturally-sourced PSA in synthesis of medicinal agents has notable shortcomings since
it is predominantly sourced from bacteria and of insufficient purity for drug development. The impurities
which may contaminate protein-PSA conjugates present undefined risk for acute and chronic immune
responses.22 The microheterogeneity of naturally sourced PSA additionally poses a chemical challenge22-24
since it complicates optimization of pharmacokinetics (PK) and pharmacodynamic (PD) performance.
Finally, the poly-dispersity of PSA hinders its chemical characterization and reproducibility in synthesis of
registered drugs.25-28
We hypothesized that a repeat polymer of mono-sialic acids site-specifically conjugated to a small
peptide (termed here as “sialopeptide”), could mimic the favourable features of PSA yet be accessible by a
highly defined synthetic approach to yield a single final product. Previous efforts to modify insulin by well-
characterized carbohydrates includes enzymatic semi-synthesis with dendritic sialyloligosaccharides,29 as
well as the total insulin chemical synthesis incorporating GalNAc and mono, di, and tri-mannose glycans.30
The semisynthetic glycol-insulin analogues displayed slightly extended duration in action while the
synthetic glycosylated analogues possessed enhanced stability against oligomerization and proteolysis.
Both methods provide encouragement for the identification of insulin analogues enhanced through
carbohydrate modification and prepared by straightforward synthetic methods to yield well-defined
chemical entities. We envisioned the chemical synthesis of a series of sialopeptide modified insulins in
order to systematically assess the relationship between sialic acid density and the physical and biological
properties of the resulting conjugates.
We report the synthesis of a set of sialopeptide-enhanced insulin analogues (termed as “Sialic-Ins”)
(Figure 1) with the characterization of their conformation, biophysical stability, and biological activity by
in vitro and in vivo methods. The collective results indicate that Sialic-Ins represent a novel approach to
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significantly improve peptide stability while retaining full biological potency. The subtle modification of
peptides and proteins with a single, or several sialic acids offers a compelling route to achieving superior
macromolecular drug candidates.
H
N
H
N
O
O
O
NH2
Ac
F
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H
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COOH
N H2
OH
HO
H
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H
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HN
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O H
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O H
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AcHN
T
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Chain B
O H
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E
G
A
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S
L
H
C
E
Y
V
L
L
1-4 (n = 1-4)
Figure 1. Sialopeptide-modified insulin, Sialic-Ins 1-4
Results
We select the tetrapeptide Phe-Glu-Lys-Arg representing a combination of charged, hydrophilic
and hydrophobic amino acids as a core backbone, with the sialic acid covalently attached to the side chain
amine of Lys (Figure 1). The resulting sialopeptide can be coupled repeatedly to provide a polysialic acid-
peptide mimicking PSA. The sialopeptide is acetylated at the N-terminus and extended at the C-terminus
with a Cys-amide providing a point for attachment to insulin through directed disulfide bond formation.
Human insulin can be selectively modified at the B-chain N-terminal Phe to introduce a single thiol for
conjugation with the sialopeptide C-terminal cysteine. The relatively labile disulfide bond was purposely
chosen in order to minimize immunogenic risk as in metabolic degradation it represents a chemically less
stable form of peptide attachment. Alternatively, a more stable form of attachment such as a thioether could
prove superior in certain specific applications.
O
O
O
O
O
O
O
O
O
O H
O
O H
O H
O H
O
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O H
O H
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Cl
a
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c
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Sialic Acid
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Scheme 1. Synthesis of sialic acid building block. Reagents and conditions: (a) MeOH, TFA, 25 oC, 48
h, 87% (b) Ac2O, DMAP, pyridine, 25 oC, 85% (c) AcCl, MeOH 5 oC 48 h 96% (d) AllOH, silver salicylate,
MS (4 Å), 23 oC, 4 h 82% (e) NaIO4, RuCl3XH2O, CCl4:ACN:H2O (2:2:3), 23 oC, 2 h, 80% (f) NHS, DCC,
DMF, 23oC, 2 h, used without purification.
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Acetylation is commonly employed to mask the numerous carbohydrate hydroxyl groups during
the synthesis of glycopeptides.31-34 Following the previously-established routes,35-37 we started from sialic
acid and prepared the fully-protected sialic acid building block 9 which was converted to the NHS activated
ester form 10 (Scheme 1). Separately, peptides 11a-d consisting of 1 to 4 repeats of Phe-Glu-Lys-Arg unit
and derivatized by C-terminal Cys extension and N-terminal acetylation were prepared by Fmoc (11a) or
Boc (11b-d) Chemistry-based solid-phase peptide synthesis (SPPS). The activation of Cys to
Cys(thiolpyridine) was achieved during the resin cleavage by including 2,2‘-dithioldipyridine in the TFA
cocktail (11a) or by treating the crude peptides with 2,2‘-dithioldipyridine in AcOH-containing aqueous
acetonitrile (11c-d). Peptides 11a-d were smoothly converted to the respective sialic acid-decorated forms
12a-d by acylation with the activated ester 10 (1.5 equiv per Lys residue) in DMF with isolated yields of
80-90% (Scheme 2)
It has been reported that the deacetylation conditions are often problematic for glycopeptide
synthesis, leading to β-elimination at residues such as Ser, Thr or Cys.31, 32, 34 To identify a realiable
deprotection protocol for the sialopeptides, a model peptide 13 was also prepared following a procedue
similar to the synthesis of peptides 12. Peptide 13 when treated with 40 mM sodium hydroxide (NaOH) at
4 °C for 3 h was found to completely liberate the acetyl and methyl esters to provide sialopeptide 14.
(Scheme 2). However, when the same deprotection condition was applied to the Cys(thiolpyridine)-
extended peptide 12a, the hydrolysis of the acetyl groups proceeded slowly, and prolonged treatment or
higher concentrations of NaOH caused the degradation of pyridyl disulfide bond (Scheme 2). The mixed
trial deprotection results here directed us to construct the disulfide linkage between the sialopeptide and
insulin prior to deprotection of the sialic acids.
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O
O
O
O
O
O
a
H
H
H
H
n
Solid-Phase
Peptide Synthesis
R N
F
E
N
[
F
E
N
R N
]
[
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n
NH2
NH2
S
O
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O
11a (n = 1)S
11b (n = 2)
11c (n = 3)
11d (n = 4)
O
12a (n = 1) S
12b (n = 2)
12c (n = 3)
12d (n = 4)
H2
N
O
O
O
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N
O
N
NH
H
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O
O
O
O
O
O
O
H
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F
E
N
R NH2
H
F
E
N
R NH2
O
O
O
O
O
O
O
b
O
OH
O
O
OH
O
O
OH
HO
NH
H
HO
O
NH
N
H
O
N
O
O
O
O
O
H
O
H
N
O
O
F
E
R N
NH2
13
14
SH
O
O
O
O
O
O
OH
O
c
O
O
12a
15
NH
H
N
O
O
O
O
d
Human
Insulin
e
H
f
H
N
O
O
TrtS
N
HS
TrtS
+
Acrolein
16
17
18
O
O
O
O
O
O
H
H
n
H
N
H
[
F
E
N
R N
]
[
F
E
R N
]
n
NH2
NH2
S
S
S
S
O
h
O
g
OH
O
OH
O
O
O
O
O
O
O
H
N
OH
HO
H
N
12 + 18
O
HO
O
NH
H
N
NH
O
H
N
O
O
O
O
19a (n = 1)
19b (n = 2)
19c (n = 3)
19d (n = 4)
1 (n = 1)
2 (n = 2)
3 (n = 3)
4 (n = 4)
O
O
Scheme 2. Synthesis of Sialic-Ins 1-4. Reagents and conditions: (a) 10, DIEA, DMF, 1 h, 83-94%; (b) 40
mM NaOH, ACN/H2O (3:1), 3 h; (c) 40 mM NaOH, ACN/H2O (3:1), 12 h; (d) Triphenylthiomethane,
Et3N, DCM, 1h, 90%; (e) NaCNBH3 25 mM acetate buffer (pH 4.0), 6 h, 75%; (f) TFA, TIPS, H2O, 5 min,
93%; (g) 6.0 M GnHCl buffer, total peptide concentration 20 mg/mL, 1 h, 62-85%; (h) 15% NH4OH, 0-
4C, 6-24 h, quant.
The single N-terminal insulin thiol was installed by reductive amination with Trt-S-CH2CH2CHO
16 which was prepared from triphenylthiomethane and acrolein (Scheme 2). The reaction was conducted
in a mixed solvent of acetonitrile and water at pH 4.5 with sodium cyanoborohydride. The resulting insulin
conjugate 17 was treated with trifluoroacetic acid (TFA), TIPS and H2O to remove the Trt group and
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provide thiopropyl-insulin 18 in 70% yield from human insulin. The attachment of the linker to the B1
position was unequivocally confirmed through endoprotease Glu-C mapping (Figures S9-10). The ligation
of equal molar amounts of protected sialopeptides 12a-d and thiopropyl insulin 18 was conducted in 6.0 M
guanidine hydrochloride solution at pH 6.0 and 25 °C at a total peptide concentration of 20 mg/mL. Under
such conditions, the reaction was typically completed within 1 h to provide Sialic-Ins 19a-d as protected
form in 70-85% isolated yields (Figures S11-14). Diluted NaOH treatment of Sialic-Ins 19a resulted in a
complex mixture of deprotected, and chemically degraded insulin derivatives. An alternative treatment with
aqueous ammonium hydroxide (14-15% aq) at 4°C was found to completely remove the acetyl and methyl
ester while preserving the insulin structure. All analogs 1-4 of Sialic-Ins were prepared by this method in
nearly quantitative isolated step yields (Figures S15-18). The overall synthetic yield of each Sialic-Ins 1-
4 was 50-60% from rDNA-derived human insulin.
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Far-UV CD was employed to evaluate the impact of the sialopeptides upon insulin higher-order
physical structure. The spectra of Sialic-Ins 1, 2, and 3 were similar to that of the human insulin implying
the attachment of multiple sialic acids does not appreciably alter insulin structure. Interestingly, Sialic-Ins
4, with four sialic acid moieties revealed a slightly altered spectrum, suggesting that there is a limit to the
total sialic acid content that can be accommodated (Figure 2A)
Table 1. In vitro activity as determined by insulin receptor B autophosphorylation in three separate
experiments.
EC50 (nM)
STDev (±)
0.079
Insulin
0.315
Sialic-Ins 1 0.200
Sialic-Ins 2 0.354
Sialic-Ins 3 0.378
Sialic-Ins 4 1.560
0.070
0.096
0.165
0.128
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The in vitro bioactivity of the Sialic-Ins analogs was assessed by their ability to stimulate the
autophosphorylation of the insulin receptor isoform B in cells that over-express it. All Sialic-Ins analogues
with the exception of 4 preserved potency comparable to native insulin. The single analogue 4 was
approximately fivefold less potent suggesting that the subtle structural change determined by CD spectra is
diminishing biochemical signaling at the insulin receptor (Table 1).
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A
B
Insulin Aggregation
Sialic-Ins 1
Sialic-Ins 2
Sialic-Ins 3
Sialic-Ins 4
Human Ins
1000
900
800
700
600
500
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100
0
Day 1
Day 2
Day 2.5
Day 3
Day 4
Day 5
C
Figure 2. A, Circular dichroism profiles of Sialic-Ins 1-4. B, Th-T anaylsis of aliquots at days 1-5. All
samples were formulated in PBS at 1 mg/mL +/- 28 µg/mL zinc ion. The samples include PBS, Human
Insulin-zinc crystal formulated directly; Human Insulin without zinc additive; Human Insulin with zinc
additive; Sialic-Ins 1 without zinc additive; Sialic-Ins 1 with zinc additive; Sialic-Ins 3 without zinc additive;
and Sialic-Ins 3 with zinc additive. C, Changes in blood glucose concentration after subcutaneous
administration of insulin and Sialic-Ins in streptozotocin (STZ)-treated mice over 24 h. Data are expressed
as meanSEM; n=8 per group.
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The influence of the sialic acid modification on the physical stability of the resulting insulin
derivatives was determined for Sialic-Ins 1 and 3 as compared to native human insulin. Each peptide was
formulated as a 1.0 mg/mL solution in PBS at pH 7.4 with and without ZnCl2 (28 µg /mL, 3 zinc ions per
insulin hexamer). Zinc at this concentration promotes insulin hexamer and thereby protects insulin from
fibrillation.38 These peptides were subjected to thermal and mechanical stress by incubation at 37 °C and
400 rpm in an orbital shaker. Aliquots were withdrawn every 24 h for analysis by Thioflavin T (ThT) assay,
which quantifies aggregated peptide. The results indicate time to initial aggregation of human insulin
without zinc was 48 h, followed by human insulin with zinc at around 60 h (Figure 2B). Consistent with
ThT assay results, turbidity and precipitation were also visually observed for these two treatments at 48 and
60 h respectively (Figure 3). In sharp contrast, Sialic-Ins 1 and 3 formulated even without zinc appeared
physically stable through day 5 as only background signal was detected which remained unchanged as
determined by ThT assay (Figure 2B). Continuous monitoring of these four samples up to four weeks did
not identify any evidence of biophysical aggregation (Figure 3). The fact that no differences were observed
in the absence of zinc suggests that Sialic-Ins are stable in molecular forms other than a hexamer. The
possibility that they exist in monomeric form raises the potential that they are insulin analogs with a faster
and more precise time action. Furthermore, Salic-Ins 1 performed equally to Salic-Ins 3 indicating that the
enhanced stability could be achieved by incorporation of only a single sialic acid. Nevertheless, the exact
mechanism of action remains to be established by systematic structural and biophysical characterization of
Sialic-Ins 1. Additional study specifically investigating the nature of the peptide linker relative to the
carbohydrate remains a focus for continued investigation.
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Human Insulin +
Human Insulin
(zinc crystal)
Human Insulin
PBS
PBS
A-1
B-1
B-2
zinc ion
60 h
48 h
60 h
C-1
C-2
D-1
D-2
Sialic-Ins 1
Sialic-Ins 1
Sialic-Ins 3
Sialic-Ins 3
4 weeks
+ zinc ion
4 weeks
4 weeks
+ zinc ion
4 weeks
Figure 3. Photographs of insulin samples agitated at 37 °C and 400 rpm. The photo of human insulin
samples were taken at the onset of cloudiness while the ones of Sialic-Ins samples were taken at 4 weeks.
The photos include PBS; Human Insulin-zinc crystal formulated directly, 60 h; Human Insulin without
zinc additive, 48 h; and Human Insulin with zinc additive, 60 h; Sialic-Ins 1 without zinc additive; Sialic-
Ins 1 with zinc additive; Sialic-Ins 3 without zinc additive; and Sialic-Ins 3 with zinc additive.
The in vivo pharmacological effort of Sialic-Ins was assessed in a widely used mouse model of
insulin deficiency. Insulin deficiency was achieved by treating mice with streptozotocin (STZ),
a glucosamine-nitrosourea that induces selective cytotoxicity on the insulin-producing pancreatic beta-cells.
STZ-treated mice exhibited hyperglycemia (>300mg/dl) and we monitored the reduction of blood glucose
after subcutaneous injection of each of the Sialic-Ins 1-4 at 10 nmol/kg. All compounds demonstrated potent
ability to lower glucose with a time action that was largely comparable to native hormone (Figure 2C).
Specifically, the doubly substituted insulin conjugate, 2 exhibited a slight but statistically significant
extended duration of action at 2 and 3 h. The most intensively modified analog 4, which was of reduced in
vitro potency, was of full in vivo potency (with the exception of a statistically significant delayed onset at
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the initial 30 min), something that has been previously noted for other insulin analogs of comparably
reduced potency.39
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Discussion and Conclusions
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Inspired by the potential of polysialic acid and seeking to mitigate their structural complexity, a set
of sialopeptide-derivatized insulin analogs have been prepared in total yields of >50%. The core
sialopeptide unit consists of a single sialic acid containing tetrapeptide. The attachment to human insulin
of singular core sialic acid tetrapeptide and up to a total of four has little impact on structure as assessed by
CD, or bioactivity as determined by in vitro and in vivo study, but confers remarkable improvement in
biophysical stability. The presence of sugar extends the time to onset of fibrillation from 2 days for native
insulin to longer than four weeks for Sialic-Ins. Of great significance is that the formulation with zinc was
not required to achieve greater stability, and with only a single sialic acid adduct was sufficient to achieve
full enhancement. The collective results support a simple sialopeptide introduction as a powerful
technology to significantly strengthen insulin stability without compromising its structure or bioactivities.
Hossain, Wade and colleagues have recently reported the synthesis of a sialylundecasaccharide-conjugated
insulin using a large oligosaccharide complex isolated from egg yolk. The fully synthetic insulin was
modified with a single additional cysteine orthogonally protected for subsequent sugar conjugation. This
sialylundecasaccharide-conjugated insulin was equally potent to human insulin and more resistant to
fibrillation for up to one week,40 consistent with the findings reported here. Collectively, this work
illustrates the power of sialic acid-based modifications to strengthen physical properties of insulin without
altering biological function. The fact that the introduction of a single sialic acid can be employed to
significantly optimize a drug as important as insulin suggests that the methodology is likely to be of similar
value in enhancing other macromolecular drug candidates with chemistry much more defined than that
historically employed.
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Experimental Section
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Reagents and solvents were bought and used without purification unless otherwise stated. Reagents
including trifluoroacetic acid (TFA), piperidine, triisopropylsilane, (TIPS), diisopropylethyl amine
(DIPEA), N,N'-Dicyclohexylcarbodiimide (DCC), sodium cyanoborohydride( NaBH3CN), allyl alcohol
(AllOH), acetyl chloride (AcCl), sodium periodate (NaIO4), ruthenium (III) chloride hydrate (RuCl3.XH2O),
N-Hydroxysuccinimide (NHS), carbon tetrachloride (CCl4), acetic anhydride (Ac2O), guanidine
hydrochloride (GnHCl), 2,2′-Dithiodipyridine (DTDP), and solvents including methanol (MeOH),
acetonitrile (ACN), diethyl ether, tetrahydrofuran (THF) N-methyl-2-pyrrolidone (NMP), N,N’-
dimethylforamide (DMF) and dichloromethane (DCM) were purchased from Sigma Aldrich. Fmoc-
protected amino acids including Fmoc-L-Phe, Fmoc-L-Glu(OtBu), Fmoc-L-Lys(Boc), Fmoc-L-Arg(Pbf),
and Fmoc -L-Cys(Trt), and Boc-protected amino acids including Boc-Phe, Boc-Glu(OcHx), Boc-Lys(Cl-
Z) , Boc-Arg(Tos) and Boc-Cys(MeBzl) were purchased from Midwest Bio-Tech Inc. LC-MS analysis was
conducted on Agilent 1260 infinity LC system coupled to an Agilent 6120 quadrupole mass spectrometer
with a analytical RP-HPLC column Phenomenex Kinetex C8 2.6 µm 100 Å (75 x 4.5 mm) using eluent
gradient consisting of 10-80% B over 10 min (Buffer A: 0.05% TFA in H2O; buffer B: 0.05% TFA in 90%
aqueous ACN). The crude peptides were purified using preparative RP-HPLC system which employed a
2.0 x 25 cm Phenomenex C18 column in a 0.1% TFA buffer and an increasing ACN gradient. UV
absorbance was measured at wavelengths 220 and 230 nm via Waters dual wavelength detector 2487.
Purified fractions were collected at a flow rate of 8-10mL/min Prostar model 701 fractions collector.
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The synthesis of sialic acid derivative was monitored using think layer chromatography (TLC) technique
with Silica Gel HLTLC Plates (w/UV254, 250μm, 20 x 20 cm) from Sorbent Technologies. The
chromatograms were developed, and TLC plates were visualized by dipping them in sulfuric acid dip
followed by charring at 150 °C and their Rf values determined. Purification of all products was done using
a medium pressure liquid chromatography (MPLC) system Teledyne ISCO (Teledyne Technologies
Company, 4700 Superior St., CA, www.isco.com). The purity of all non-peptidic compounds were
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determined by NMR to be > 95%. H and C NMR spectra were obtained using on a Varian VXR 400
(400 MHz/ 101 MHz), Varian INOVA 400 (400 MHz/ 101 MHz), Varian 500 (500 MHz/ 125 MHz) or
Varian 600 (600MHz) instruments. Chemical shift values are measured and reported in parts per million
(ppm) on the δ scale. Coupling constants and signal splitting patterns were recorded as J values in Hz.
Abbreviations for multiplicities such as s = singlet, d = doublet, dd =double of doublet, t = triplet, q =
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quartet, m = multiplet, bs = broad singlet were used. H NMR and 13C NMR spectra taken for compounds
1
in CDCl3 were referenced to the solvent peak at 7.260 ppm (1H) and 77.16 ppm (13C). On the other hands,
those taken in CD3OD were reported relative solvent peaks at 3.31 ppm (13H) and 49.00 ppm (13C).
Methyl 5-Acetamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosonate (5). To a solution of N-
acetalneuraminic acid (20.0 g, 64.7 mmol) in anhydrous methanol (400 mL) was added trifluoroacetic acid
o
o
(0.2 mL) at 23 C. The reaction mixture was allowed to stir at 23 C for 48 h, after which TLC analysis
TLC (EtOAc/iPrOH/H2O: 2:2:1), confirmed the formation of the product. The reaction mixture was filtered
over a pad of celite, concentrated and dried under vacuum. The dried material was dissolved in anhydrous
pyridine and filtered via medium and the residue was washed several times with methanol. The filtrate was
concentrated under reduced pressure to give the product, 5 as a white solid (18.2 g, 87%), whose data
matched the reported ones.35
Methyl 5-Acetamido-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosonate
(6). To a solution of 5 (5.00 g, 15.5 mmol) in anhydrous pyridine (30 mL) was added acetic anhydride
(11.8 g, 116 mmol) and catalytic amount of DMAP. The reaction mixture was at room temperature for 12
h after which TLC (acetone: toluene: 1:1) analysis confirmed the formation of the product. The reaction
mixture was poured into a crashed ice and stirred until all the ice melted. The aqueous mixture was extracted
with EtOAc (3x50 mL). The combined organic layers was washed with saturated solution of ammonium
chloride, dried, filtered, concentrated under vacuum to give the crude, which was purified via MPLC using
ISCO (using 0-50% acetone in toluene) to give the product, 6 (7.01 g, 85%), whose data agreed with the
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literature report.35 1H NMR (500 MHz, CDCl3) δ 5.78 – 5.64 (m, 1H), 5.33 (dd, J = 4.8, 1.5 Hz, 1H), 5.26
– 5.11 (m, 1H), 5.00 (ddd, J = 7.2, 4.8, 2.5 Hz, 1H), 4.45 (dd, J = 12.4, 2.6 Hz, 1H), 4.20 – 3.96 (m, 3H),
3.73 (s, 3H), 2.48 (dd, J = 13.4, 4.9 Hz, 1H), 2.09 (d, J = 2.4 Hz, 6H), 2.00 (s, 3H), 1.97 (d, J = 1.1 Hz, 6H),
1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 170.89, 170.66, 170.53, 170.40, 170.32, 170.28, 170.19,
170.08, 170.04, 169.77, 169.65, 169.04, 168.60, 168.25, 168.21, 167.88, 166.31, 164.60, 97.45, 77.35,
77.30, 77.10, 76.85, 72.80, 71.49, 68.38, 68.36, 67.77, 62.11, 53.14, 49.09, 35.90, 23.06, 20.85, 20.84,
20.80, 20.77, 20.73, 20.72, 20.67, 10.56.
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Methyl
5-Acetamido-2-chloro-4,7,8,9-penta-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-2-
nonulopyranosonate (7). A solution of 6 (2.00 g; 4.07 mmol) in a mixture of AcCl and HOAc in the ratio
of 1:1 (40 mL) was saturated at 0 oC with anhydrous HCl(g) generated by addition of concentrated H2SO4
to anhydrous calcium chloride. The resulting mixture was stirred at 23 oC for 24 h, after which TLC analysis
indicated complete formation of the chloride, which was concentrated under reduced pressure followed by
co-evaporation with toluene three times to give a white foam as the product 7 (1.98 g; 3.88 mmol; 96%),
which was taken to the next step without purification.35
Methyl
(5-acetamido-2-allyl-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-
nonulopyranosid)onate (8). The chloride 7 (1.98 g; 3.88 mmol) was dissolved in allyl alcohol (21.6 mL;
318 mmol; 82 equiv) followed by the addition of silver salicylate (1.43 g; 5.82 mmol; 1.5 equiv) resulting
in a suspension, that was stirred at 23 oC in the dark until TLC analysis confirmed product formation (2 h).
The reaction mixture was filtered over a pad celite and the precipitate washed with chloroform. The filtrate
was washed with cold sat. NaHCO3 solution, 5% Na2S2O3 and water. The organic layer was dried (Na2SO4),
filtered, concentrated under reduced pressure to give the crude, which was purified via MPLC ISCO to
afford the product 8 (1.69 g; 82%) whose data was in agreement with the published one.36, 37 1H NMR (500
MHz, CDCl3) δ 7.26 (s, 1H), 5.95 – 5.76 (m, 1H), 5.44 – 5.22 (m, 4H), 5.22 – 5.11 (m, 2H), 4.86 (ddd, J
= 12.1, 9.5, 4.6 Hz, 1H), 4.34 – 4.22 (m, 2H), 4.09 (td, J = 13.1, 12.6, 7.7 Hz, 3H), 3.87 (ddt, J = 12.9, 5.8,
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1.5 Hz, 1H), 3.78 (s, 3H), 2.61 (dd, J = 12.8, 4.6 Hz, 1H), 2.14 (d, J = 6.4 Hz, 6H), 2.03 (d, J = 6.8 Hz, 6H),
1.87 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 170.95, 170.61, 170.22, 170.09, 168.33, 133.52, 117.22, 98.45,
77.33, 77.28, 77.08, 76.82, 72.52, 69.12, 68.73, 67.36, 65.85, 62.40, 52.62, 49.27, 38.01, 23.11, 21.08,
20.82, 20.80, 20.73, 20.68.
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Methyl (2-Carboxylmethyl-5-acetamido-4,7,8,9-tetra-O- acetyl-3,5-dideoxy-α-D-glycero-D-galacto-
2-nonulopyranosid)onate (9). To a biphasic solution of the allyl product, 8 (500 mg; 0.941 mmol; 1.0
equiv) and NaIO4 (800 mg; 3.76 mmol; 4 equiv) in CCl4 (2 mL) acetonitrile (2 mL) and water (3 mL) was
added RuCl3.XH2O (9.76 mg; 0.0471 mmol; 0.05 equiv). The reaction solution was stirred vigorously at 23
oC for 2 h and then diluted with CH2Cl2 (30 mL) followed by the separation and extraction of the aqueous
layer with CH2Cl2 (3 x 30 mL). The combined organic layer was washed with H2O, dried over anhydrous
Na2SO4, filtered, concentrated and purified with MPLC (50-100% EtOAc/CH2Cl2) to give the product 9
(412 mg; 80%). The NMR agreed with the literally reported ones.36 1H NMR (500 MHz, CDCl3) δ 7.26
(d, J = 1.0 Hz, 1H), 5.37 (ddt, J = 8.8, 5.8, 2.3 Hz, 2H), 5.29 (dd, J = 8.4, 1.5 Hz, 1H), 4.96 (ddd, J = 11.8,
9.4, 4.7 Hz, 1H), 4.35 (d, J = 16.7 Hz, 1H), 4.32 – 4.19 (m, 2H), 4.14 – 3.94 (m, 3H), 3.80 (s, 3H), 2.70
13
(dd, J = 13.0, 4.7 Hz, 1H), 2.19 – 1.94 (m, 14H), 1.89 (s, 3H). C NMR (126 MHz, CDCl3) δ 171.74,
171.02, 170.80, 170.70, 170.28, 170.18, 167.59, 98.27, 77.27, 77.22, 77.02, 76.77, 72.55, 68.91, 68.45,
67.17, 62.41, 61.68, 53.12, 49.49, 37.38, 23.11, 21.09, 20.84, 20.76, 20.73.
Methyl 2-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethoxy-5-acetamido-4,7,8,9-tetra-O- acetyl-3,5-
dideoxy-α-D-glycero-D-galacto-2-nonulopyranosid)onate (10). To a solution of the acid 9 (1.15 g; 2.09
mmol; 1.0 equiv) in anhydrous DMF (15 mL) under argon were added NHS (0.240 g; 2.09 mmol, 1.0
o
equiv) and DCC (0.43 g; 2.09 mmol; 1.0 equiv). The resulting solution was stirred at 23 C until TLC
analysis showed a lower running spot corresponding to a complete transformation of the starting material
in the desired product 10. The reaction mixture was kept and used as stock solution for the subsequent steps.
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Fmoc-based Synthesis of Peptide 11a
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20% piperidine in DMF (10 mL) was added to the Polystyrene AM RAM resin (0.685 g, 0.50 mmol), the
resulting mixture was shaken for 10 min on a rotator and then drained. This step was repeated one time.
The Fmoc-deprotected resin was then washed with DMF (6 x 10 mL) followed by the addition Fmoc-
Cys(Trt)-OH (1.464 g, 2.5 mmol, 5 equiv), DEPBT (0.748 g, 2.5 mmol, 5 equiv), DMF (10 mL) and
DIPEA (0.646 g, 5 mmol, 10 equiv). The mixture was agitated on a rotator for 2 h. The resin was then
drained and washed with DMF (3 X 10 mL), followed by the treatment of 20% piperidine in DMF (10 mL)
for 10 min twice. The following couplings of Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-
OH, Fmoc-Phe-OH were conducted following the same protocol. The resulting resin was then treated with
Ac2O (1.021 g, 10 mmol, 20 equiv.) in DMF (10 mL) for 2 h followed by washing with DMF and DCM.
To cleave and activate the pentapeptide from the resin, DTDP (1.102 g, 5 mmol), TIPS (0.5 mL), H2O (0.5
mL) and TFA (19 mL) were added to the resin, and the resulting mixture was agitated for 2 h followed by
filtration and rotavapor to remove TFA. The resulting material was treated and washed with cold diethyl
ether, dissolved in ACN/H2O and purified by prep. RP-HPLC to give the pentapeptide 11a 115 mg in a
yield of 28% and purity by analytical HPLC of 99%. M.W. calculated: 832.0; observed: 831.4. (Scheme
S1 and Figure S1)
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Boc-based Synthesis of Peptides 11b-11d
0.2 mmol of 4-methyl benzhydrylamine resin (MBHA) was placed in a CSBio reaction vessel and 2 mmol
tubes of Boc amino acids were aligned on the AA wheel to correspond to the desired sequence. 2.0 mmol
acetic acid was placed in the last tube. 30 min couplings were run using 0.5M DEPBT and DIPEA in DMF
on a CSBio synthesizer. At the completion of the chain assembly, the acetylated peptide resin was
transferred to an HF reaction vessel after washing with DMF and DCM. The HF reaction vessel was
attached to the HF apparatus, cooled in a dry ice/methanol bath, evacuated, and 5-7 ml of liquid hydrogen
fluoride was condensed in. The reaction was stirred 60 min in an ice bath then the HF was removed under
vacuum. The residue was suspended in ethyl ether (20-30 ml) and the product/resin suspension was filtered
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and washed with ether. The peptide was extracted into 2% aqueous acetic acid/20% acetonitrile and S-
pyridyl activation was achieved by the addition of DTDP (2 mmol) for 4 h. The resulting peptide was
purified by prep. PR-HPLC followed by freeze-drying (Scheme S2).
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Peptide 11b. The above described general procedure was followed to synthesize peptide 11b at 0.2 mmol
scale which after prep. RP-HPLC purification offered 109 mg of product at 39% yield based on the resin
substitution. Purity by analytical HPLC: 90%; M.W. calculated: 1392.7; observed: 1392.7 (Figure S2).
Peptide 11c. The above described general procedure was followed to synthesize peptide 11c at 0.2 mmol
scale which after prep. RP-HPLC purification offered 147 mg of product at 38% yield based on the resin
substitution. Purity by analytical HPLC: 99%; M.W. calculated: 1952.3; observed: 1953.0 (Figure S3).
Peptide 11d. The above described general procedure was followed to synthesize peptide 11d at 0.2 mmol
scale which after prep. RP-HPLC purification offered 151 mg of product at 30% yield based on the resin
substitution. Purity by analytical HPLC: 98%; M.W. calculated: 2513.9; observed: 2513.4 (Figure S4).
General procedure for preparing Sialopeptides 12a-d. The thiolpyridine peptides (11a-d) (1.0 equiv)
was mixed with the activated sialic acid 10 (1.5 equiv per Lys) in anhydrous DMF under argon atmosphere.
The reaction was stirred at rt until LC-MS analysis confirmed the complete conversion (1 h), and then
diluted with 0.05%TFA/H2O and purified by prep. RP-HPLC to give the products.
Peptide 12a. The above described general procedure was followed to synthesize sialopeptide 12a from 11a
which after prep. RP-HPLC purification offered 68 mg of product at 89% step yield. Purity by analytical
HPLC: 98%; M.W. calculated: 1363.5; observed: 1362.6 (Figure S5).
Peptide 12b. The above described general procedure was followed to synthesize sialopeptide 12b from 11b
which after prep. RP-HPLC purification offered 61 mg of product at 83% step yield. Purity by analytical
HPLC: 93%; M.W. calculated: 2455.6; observed: 2455.0 (Figure S6).
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Peptide 12c. The above described general procedure was followed to synthesize sialopeptide 12c from 11c
which after prep. RP-HPLC purification offered 85 mg of product at 94% step yield. Purity by analytical
HPLC: 92%; M.W. calculated: 3547.7; observed: 3546.6. (Figure S7).
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Peptide 12d. The above described general procedure was followed to synthesize sialopeptide 12d from 11c
which after prep. RP-HPLC purification offered 78 mg of product at 85% step yield. Purity by analytical
HPLC: 90%; M.W. calculated: 4639.8; observed: 4638.9. (Figure S8).
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Synthesis of 3-(tritylthio)propanal 16
Triphenylthiomethane (10.0 g, 36.2 mmol, 8.3 equiv.) in anhydrous CH2Cl2 (80mL) was added to a solution
of Et3N (7.56 mL, 54.3 mmol, 12.5 equiv.) and acrolein (3.44 mL, 4.36 mmol, 1.0 equiv.). The reaction
mixture was stirred at room temperature until TLC analysis confirmed the product, 3-tritylthiopropanal (1
h). The reaction mixture was concentrated under reduced pressure to give the crude 3-tritylthiopropanal
which was recrystallized from ethyl acetate/hexane to obtain the pure product in 90% yield. 1H NMR (500
MHz, CDCl3) δ 9.56 (t, J = 1.3 Hz, 1H), 7.54 – 7.37 (m, 7H), 7.39 – 7.12 (m, 11H), 2.48 (td, J = 7.0, 0.9
Hz, 3H), 2.37 (tt, J = 7.1, 1.1 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 200.29, 144.54, 129.56, 128.00,
126.79, 77.32, 77.07, 76.81, 67.02, 42.69, 24.44.
Synthesis of 3-Thiopropyl Insulin 17
Human insulin (100 mg, 0.0172 mmol) was dissolved in 10 mM HCl (10 mL) and the solution neutralized
with 10 mM NaOH (10 mL). To the resulting solution were added acetonitrile (25 mL), 25 mM NaOAc
butffer (25 mL pH 4.0), 3-(tritylthio)propanal (5.73 mg, 0.0272 mmol, 1.0 equiv) and NaCNBH3 (67.9 mg,
1.08 mmol, 20 equiv). The reaction was monitored with LC-MS at 30 min intervals. After 4 h, another 3-
(tritylthio)propanal (5.73 mg, 0.0272 mmol, 1.0 equiv) was added and the reaction allowed to proceed until
LC-MS analysis confirmed conversion of most of the insulin. The reaction mixture was diluted with buffer
A containing 0.05% TFA in 10% acetonitrile/water. The mixture was purified via reverse phase
chromatography using a 2.0x25cm Phenomenex C18 column in a 0.1%TFA buffer and an increasing
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acetonitrile gradient to give the product 17 after freeze-drying (76 mg, 75%). Purity by analytical HPLC:
97%; M.W. calculated: 6124.5; observed: 6123.6 (Figure S9). Endoprotease Glu-C digest. 0.2 mg of 17
was dissolved in 0.5 mL buffer (25 mM NH4HCO3, pH 8.0) and 20 µL of GluC (Thermo Scientific, 20 µg
dissolved in 500µL of 25 mM NH4HCO3 buffer, pH 8.0) was added to the solution. The reaction was
incubated overnight at 37 °C. Reaction was analyzed by analytical LCMS (10-80% MeCN gradient over
10 min., 0.05% TFA, Kinetex C8/ 2.6µ/100A/ 75x4.60 mm column) (Figure S9).
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Synthesis of 3-Thiopropyl Insulin 18
3-(Tritylthio)propyl insulin 17 (80.0 mg, 0.013 mmol) was dissolved in trifluoroacetic acid (TFA)
containing triisopropylsilane (2%) and H2O (2%) at 7.5 mg/mL concentration of the insulin derivative.
After 5 mins, detritylated material was recovered by cold ether precipitation and centrifugation. The
recovered material was washed three times with cold ether. LC-MS analysis confirmed that detritylation
was quantitative. The material was dissolved in H2O and freeze-dried directly without RP-HPLC
purification to provide 71 mg 18 with 93% step yield. Purity by analytical HPLC: 99%; M.W. calculated:
5881.7; observed: 5881.8 (Figure S10).
General method of synthesizing protected Sialic-Ins 19a-d. The freeze-dried powder of 2-thiopyridyl
activated sialopeptide (12a-d) (1.0 equiv) and thiopropyl insulin 18 (1.0 equiv) were combined in a 20 mL
vial followed by the addition of 6.0 M guanidine hydrochloride buffer, pH 6.0 resulting in a reaction
solution of total peptide concentration of 20.0 mg/mL. The reaction was stirred at rt until LC-MS analysis
confirmed the complete conversion (60 min). The reaction mixture was then diluted with 0.05%TFA/H2O
and purified via prep. RP-HPLC to give the products 19a-d after freeze-drying.
Sialic-Ins 19a. Sialopeptide 12a (17.7 mg, 12.9 µmol) and thiopropyl insulin 18 (76.3 mg, 12.9 µmol) were
ligated according to the above described general ligation procedure which after purification by prep. RP-
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HPLC provided 65 mg protected Sialic-Ins 13 with 70% step yield Purity by analytical HPLC: 95%. M.W.
calculated: 7134.5; observed: 7134.0 (Figure S11).
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Sialic-Ins 19b. Sialopeptide 12b (27.6 mg, 11.2 µmol) and thiopropyl insulin 18 (66.1 mg, 11.2 µmol) were
ligated together according to the above described general ligation procedure which after purification by
prep. RP-HPLC provided 56 mg protected Sialic-Ins 14 with 62% step yield. Purity by analytical HPLC:
95%. M.W. calculated: 8226.6; observed: 8226.5 (Figure S12).
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Sialic-Ins 19c. Sialopeptide 12c (42.6 mg, 11.4 µmol) and thiopropyl insulin 18 (67 mg, 11.4 µmol) were
ligated together according to the above described general ligation procedure which after purification by
prep. RP-HPLC provided 91 mg protected Sialic-Ins 15 with 82% step yield. Purity by analytical HPLC:
95%. M.W. calculated: 9318.7; observed: 9319.0 (Figure S13).
Sialic-Ins 19d. Sialopeptide 12d (40.2 mg, 8.67 µmol) and thiopropyl insulin 18 (51 mg, 8.67 µmol) were
ligated together according to the above described general ligation procedure which after purification by
prep. RP-HPLC provided 76 mg protected Sialic-Ins 16 with 85% step yield. Purity by analytical HPLC:
94%. M.W. calculated: 10410.8; observed: 10410.0 (Figure S14).
General method of synthesizing Sialic-Ins 1-4. The sialic acid-protected Sialic-Ins (19a-d) was placed in
a 20 mL vial at 0o C. To this was added pre-cooled solution 14-15% NH4OH. The reaction was taken to and
kept at 4 oC until LC-MS analysis revealed the complete hydrolysis of the acetate and methyl ester groups.
The reaction mixture was neutralized with 0.1 M AcOH and diluted with 0.05% TFA/H2O and purified via
prep. RP-HPLC to give the products (1-4) after freeze-drying.
Sialic-Ins 1. The protected Sialic-Ins 19a (20 mg, 2.80 µmol) was converted to Sialic-Ins 1 following the
above described general hydrolysis condition providing 18 mg of 1 with step yield of 90%. Purity by
analytical HPLC: 96%. M.W. calculated: 6952.3; observed: 6950.8 (Figure S15).
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Sialic-Ins 2. The protected Sialic-Ins 19b (21.3 mg, 2.59 µmol) was converted to Sialic-Ins 2 following the
above described general hydrolysis condition providing 19.3 mg of 1 with step yield of 95%. Purity by
analytical HPLC: 94%. M.W. calculated: 7862.2; observed: 7860.6 (Figure S16).
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Sialic-Ins 3. The protected Sialic-Ins 19c (24 mg, 2.58 µmol) was converted to Sialic-Ins 3 following the
above described general hydrolysis condition providing 22 mg of 3 with step yield of 97%. Purity by
analytical HPLC: 97%. M.W. calculated: 8772.2; observed: 8769.5(Figure S17).
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Sialic-Ins 4. The protected Sialic-Ins 19d (17.6 mg, 1.69 µmol) was converted to Sialic-Ins 4 following the
above described general hydrolysis condition providing 16.1 mg of 4 with step yield of 98%. Purity by
analytical HPLC: 97%. M.W. calculated: 9682.1; observed: 9680 (Figure S18).
Analysis of Sialic-Ins by CD Spectrometer
Circular dichroism. CD spectra were measured at 25 °C using a J-715 spectropolarimeter (Jasco, Tokyo,
Japan) in a quartz cell of 10mm path length. Spectra were the average of 10 assessments over the λ = 190–
370 nm range at 0.1 nm interval. Samples (~0.25 mg/mL) were prepared in PBS, pH 7.4. The CD results
are presented as the mean residue molar ellipticity (Θ) in deg cm2/dmol.
Aggregation Study
Lyophilized insulin derivatives were formulated in PBS at concentration of 1 mg/mL, pH 7.4, with or
without the additional ZnCl2. Triplicate samples were prepared for each insulin or insulin derivatives. All
samples were incubated at 37 C and 400 rpm in a MaxQTM 4000 Benchtop Orbital Shaker. Aliquots (20
uL) were taken every 24 h and analyzed by Thioflavin T (ThT) assay, which measures changes in
fluorescence intensity of ThT upon binding of the aggreated protein and following the modified Thioflavin-
T fluorescence assay protocol (http://www.assay-protocol.com/biochemistry/protein-fibrils/thioflavin-t-
spectroscopic-assay). 8 mg of Thioflavin-T (ThT) was dissolved in 10 mL of phosphate buffer (50 mM
sodium phosphate, 150 mM of sodium chloride, pH 7.4). Solution was filtered through 0.22 µm syringe
filter and stored at 4 C in dark. Prior to experiment the 0.3 mL of ThT stock solution was further diluted
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in 15 mL of the phosphate buffer. 5 µL of investigated peptide solution at 1 mg/mL was added to 400 µL
of working solution of ThT in phosphate buffer. Solution was incubated for 20 to 30 min. Then fluorescence
intensity was measured on Perkin-Elmer LS50B Luminescence Spectrometer (Perkin-Elmer, Waltham, MA)
with following experimental parameters: 350 µL of peptide/ThT solution in sub-micro quartz cuvette [3 x
3 x 3 mm / Z = 9.85 (Hellna GmnG & Co. KG, Mullheim, DE)] excitation  = 450 nm (slit width 5nm);
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emission  = 482 nm (slit width 10 nm) integration 10 second. Signal was averaged from 4 consecutive
points.
In vitro Characterization of Sialic-Ins 1-4 by Insulin Receptor Phosphorylation Assay
To measure receptor phosphorylation, human insulin B receptor transfected HEK293 cells were plated in
96 well tissue culture plates (Costar #3596, Cambridge, MA) and cultured in Dulbecco’s modified Eagle
medium (DMEM) supplemented with 100 IU/ml penicillin, 100 ug/ml streptomycin, 10 mM HEPES and
0.25% bovine growth serum (HyClone SH30541, Logan, UT) for 16-20 h at 37oC, 5% CO2 and 90%
humidity. Serial dilutions of recombinant human insulin or insulin analogs were prepared in duplicate in
DMEM supplemented with 0.5% bovine serum albumin (Roche Applied Science #100350, Indianapolis,
IN) and added to the wells with adherent cells. After 15 min incubation at 37 C in humidified atmosphere
with 5% CO2 the cells were fixed with 5% paraformaldehyde for 20 min at room temperature, permeabilized
with 0.1% Triton-X100 for 25 min with 5 solution changes and blocked with 2% bovine serum albumin in
PBS for 1 h. The plate was then washed three times with PBS pH 7.4 supplemented with 0.1% Tween-20
and filled with horseradish peroxidase-conjugated antibody against phosphotyrosine (Upstate
biotechnology #16-105, Temecula, CA) at manufacturer’s recommended dilution. After 3 h incubation at
room temperature the plate was washed 4 times and 0.1 ml of TMB single solution substrate (Invitrogen,
#00-2023, Carlbad, CA) was added to each well. Color development was stopped 5 min later by adding
0.05 ml 1N HCl. Absorbance at 450 nm was measured on Titertek Multiscan MCC340 (ThermoFisher,
Pittsburgh, PA). Absorbance vs peptide concentration dose response curves were plotted and EC50 values
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were determined by using logistic nonlinear 3 parameter regression in GraphPad Prism 6 (GraphPad
Software, La Jolla, CA). Each experiment was repeated at least three times.
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Acute Insulin Tolerance Test in Mice by Subcutaneous Injection
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All studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University
of Cincinnati in accordance with the US National Institutes of Health Guide for the Care and Use of
Laboratory Animals.
Male, 14 week-old, C57Bl6/J mice (Jackson Laboratories, ME) were housed on a 12 : 12 hours light-dark
cycle (7 am - 7 pm lights on) at 22 °C and constant humidity with free access to standard chow (Teklad
LM-485) and water. Mice were fasted overnight and received an injection of streptozotozin (STZ,
150mg/kg, ip, Sigma-Aldrich, MO) freshly dissolved in sodium citrate buffer (pH = 4.5), within the first 2
h following the onset of the light phase. After the injection, food was returned to the mice, and they
additionally had access to a 10% sucrose solution for 72 h. Blood glucose was measured via tail laceration
using a handheld glucometer (Freestyle, Abbot, IL) to confirm hyperglycemia. On the day of the study, the
mice (BW=20.9±1.5 g) were deprived of food at the onset of the light phase. 3 h later, and they received a
subcutaneous injection (100 µl) of vehicle (sterile, pyrogen-free phosphate buffer saline or different doses
of the compounds indicated in the results section). The blood glucose was monitored 0.5, 1, 2, 3, 6 and 24
h post injection. The statistical analysis of the results obtained in the in vivo experiments was performed
using Prism 8.3 h (GraphPad Software, CA) applying 2-way ANOVA for repeated measurements, followed
by Dunnett’s tests for multiple comparisons of the different treatments, using the human insulin group as
control. P values lower than 0.05 were considered significant. The results are presented as means ± SEM
of eight replicates per group.
Supporting Information Availability
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Fmoc chemistry-based solid phase synthesis of peptide 11a and Boc chemistry-based SPPS of peptides
11b-d; UPLC and MS spectrums of peptides 11a-d, 12a-d, 17, 18, 19a-d and 1-4.
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Crossponding Author Information:
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Prof. Nicola L. B. Pohl
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
email: npohl@indiana.edu
Dr. Fa Liu
Novo Nordisk Research Center, 530 Fairview Avenue North, Seattle, Washington 98109, USA
email: falx@novonordisk.com
Acknowledgments
This work was supported in part by the Joan and Marvin Carmack Fund. A special thanks to Mr. Jay Levy,
Drs. Kishore Thalluri, Alexander N. Zaykov, Fangzhou Wu, Fa Zhang, Yang Xu, Sontag Chris, and Piotr
Mroz in Prof. DiMarchi’s Research Laboratory at Indiana University for their support in the synthesis,
purification and characterization of the insulin analogues.
Abbreviations Used:
SPPS, solid-phase peptide synthesis; Sialic-Ins, sialic acid-conjugated human insulin; STZ, streptozotocin;
PSA, polysialic acids; rDNA, recombinant DNA; CD, circular dichroism; ThT, Thioflavin T.
Conflict of interest
V.M.G. and F.L. are currently full-time employees and shareholders of Novo Nordisk.
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