Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Nov 12;51(46):11576-84.
doi: 10.1002/anie.201206090. Epub 2012 Sep 25.

At last: erythropoietin as a single glycoform

Ping Wang  1 , Suwei Dong, John A Brailsford, Karthik Iyer, Steven D Townsend, Qiang Zhang, Ronald C Hendrickson, JaeHung Shieh, Malcolm A S Moore, Samuel J Danishefsky
Affiliations

At last: erythropoietin as a single glycoform

Ping Wang et al. Angew Chem Int Ed Engl. .
No abstract available

PubMed Disclaimer

Figures

Figure 1
Figure 1
Ribbon structure of erythropoietin containing a consensus sequence of N-linked carbohydrate domains.
Figure 2
Figure 2
New Methods for the synthesis of proteins and glycoproteins. GP = glycopeptide.
Figure 3
Figure 3
Earlier synthetic routes from our laboratory toward EPO.
Figure 4
Figure 4
Retrosynthetic strategy toward EPO.
Figure 5
Figure 5
(A) Top-down Mass Spectra of unfolded EPO. Calcd for C899H1452N242O297S5: 20562.0147 Da (average isotopes); observed: [M+12H]12+ m/z = 1715.8001, [M+14H]14+ m/z = 1582.7384, [M+15H]15+ m/z = 1371.7734, [M+16H]16+ m/z = 1286.0381, [M+17H]17+ m/z = 1210.6258, [M+18H]18+ m/z = 1143.4804, [M+19H]19+ m/z = 1083.2441, [M+20H]20+ m/z = 1029.0825, [M+21H]21+ m/z = 980.1267, [M+22H]22+ m/z = 935.7577, measured for C899H1452N242O297S5: (20562.1934± 0.1787) Da. (B) LC trace (UV) of folded erythropoietin chitobiose, and Top-down Mass Spectra of folded EPO. Calcd for C899H1448N242O297S5: 20557.9829 Da (average isotopes); observed: [M+11H]11+ m/z = 1869.8790, [M+12H]12+ m/z = 1714.2245, [M+13H]13+ m/z = 1582.3602, [M+14H]14+ m/z = 1469.4066, [M+15H]15+ m/z = 1371.5131, [M+16H]16+ m/z = 1285.8580, [M+17H]17+ m/z = 1210.3956, [M+18H]18+ m/z = 1143.2618, measured for C899H1448N242O297S5: (20558.3348± 0.3518) Da.
Figure 6
Figure 6
The image of human BFU-E colony. 60 ng/ml Synthetic folded EPO (1). and 20 ng/ml rhKL stimulate purified cord blood CD34 cells to form BFU-E colony after 2 weeks.
Scheme 1
Scheme 1
Synthesis of glycopeptide 6 en route to EPO Fragment IV.
Scheme 2
Scheme 2
Synthesis of EPO Fragment IV (9). Reactions and Conditions: (a) 1. 6 M Gn·HCl, 100 mM Na2HPO4, 50 mM TCEP, pH 7.3, 2 h; 2. piperidine, DMSO, 10 min, 55% (two steps); (b) 0.2 M MeONH2, 6 M Gn·HCl, 100 mM Na2HPO4, 50 mM TCEP, pH 4.0, 3 h, 75%.
Scheme 3
Scheme 3
Synthesis of EPO Fragment III (11). Reactions and conditions: (a) Chitobiose, HATU, DIPEA, DMSO; then TFA/TIS/H2O/phenol, 40%. AA = side chain protected sequence. AA = pseudoproline dipeptide. Side chain protecting groups are listed in the supporting information.
Scheme 4
Scheme 4
Synthesis of EPO Fragment II (13). Reactions and conditions: (a) Chitobiose, HATU, DIPEA, DMSO; then TFA/TIS/H2O/phenol, 54%.27 AA = side chain protected sequence. AA = pseudoproline dipeptide. Side chain protecting groups are listed in the supporting information.
Scheme 5
Scheme 5
Synthesis of EPO Fragment I (15). Reactions and conditions: (a) Chitobiose, HATU, DIPEA, DMSO; then TFA/TIS/H2O/phenol, 45%. AA = side chain protected sequence. AA = pseudoproline dipeptide. Side chain protecting groups are listed in the supporting information.
Scheme 6
Scheme 6
Synthesis of EPO primary structure (Ala1-Arg166). Reactions and conditions: (a) 6 M Gn•HCl, 300 mM Na2HPO4, 200 mM MPAA, 20 mM TCEP, pH 7.3, 18 h; then MeONH2•HCl, DTT, 3 h, 72%; (b) 6 M Gn•HCl, 300 mM Na2HPO4, 200 mM MPAA, 20 mM TCEP, pH 7.3, 12 h, 74%; (c) 6 M Gn•HCl, 200 mM Na2HPO4, Bond-Breaker®, VA-044, tBuSH, 37 °C, 5 h, 68%; (d) AgOAc, HOAc/H2O (1:1), 6 h, 62%; (e) 6 M Gn•HCl, 300 mM Na2HPO4, 200 mM MPAA, 20 mM TCEP, TFE, pH 7.3, 19 h, 73%..
Scheme 7
Scheme 7
Synthesis of folded EPO using convergent KCL. Reaction conditions: (a) 20 and 21, 6 M Gn•HCl, 150 mM Na2HPO4, 50 mM TCEP, pH 7.2, 5 h; then 16, 12 h; (b) 6 M Gn•HCl, 100 mM Na2HPO4, Bond-Breaker®, VA-044, tBuSH, 37 °C, 10 h, 54% (three steps); (c) AgOAc, 70% AcOH, 6 h, 73%; (d) folding conditions: 50 mM Tris•HCl, 40 μM CuSO4, N-lauroylsarcosine (2% w/v), pH 8.0.
Scheme 8
Scheme 8
Synthesis of non-glyco-EPO. Reaction conditions: a) Guanidine•HCl (6 M), Na2HPO4 (0.2 M), TCEP•HCl (0.02 M), MPAA (0.2 M), Bond-Breaker®, pH 7.2–7.4; b) MeONH2•HCl (0.3 M), pH 4.0, 34% yield over 2 steps; c) Guanidine•HCl (6 M), Na2HPO4 (0.2 M), TCEP•HCl (0.02 M), MPAA (0.2 M), Bond-Breaker®, pH 7.2–7.4; 33% yield; d) Guanidine•HCl (6 M), Na2HPO4 (0.2 M), TCEP•HCl (0.08 M) pH 7.0, VA-044, tBuSH, 37 °C; e) AgOAc, CH3CN/H2O (1:1) + 0.05% TFA then DTT, 45% yield over 2 steps; f) Guanidine•HCl (6 M), Na2HPO4 (0.2 M), TCEP•HCl (0.02 M), MPAA (0.2 M), Bond-Breaker®, pH 7.2–7.4, 35–40% yield; g) Tris•HCl (50 mM), CuSO4 (40 mM), N-lauroylsarcosine (2% w/v), pH 8.0.
See this image and copyright information in PMC

References

    1. Elliott S, Foote MA, Molineux G. Erythropoietins, Erythropoietic Factors, and Erythropoiesis. 2. Birkhäuser; Basel-Boston-Berlin: 2009.
    1. Sytkowski AJ. Erythropoietin. Wiley-VCH Verlag GmbH and Co; KGaG Weinheim J: 2004.
    1. Szenajch, Wcislo G, Jeong J-Y, Szczylik C, Feldman L. Biochim Biophys Acta. 2010;1806:82. - PubMed
    1. Rush RS, Derby PL, Smith DM, Merry C, Rogers G, Rohde MF, Viswanatham K. Anal Chem. 1995;67:1442. - PubMed
    1. Dwek RA. Chem Rev. 1996;96:683. - PubMed

Publication types

LinkOut - more resources