US 2010.0041872A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0041872 A1 DeFrees et al. (43) Pub. Date: Feb. 18, 2010 (54) GLYCEROL LINKED PEGYLATED SUGARS (86) PCT NO.: PCT/US07/80471 AND GLYCOPEPTDES S371 (c)(1), (76) Inventors: Shawn DeFrees, North Wales, PA (2), (4) Date: Jul. 28, 2009 (US); Xiao Zeng, Warrington, PA Related U.S. Application Data (US) (60) Provisional application No. 60/828,208, filed on Oct. Correspondence Address: 4, 2006. LEYDIG VOIT & MAYER, LTD Publication Classification TWO PRUDENTIAL PLAZA, SUITE 4900, 180 (51) Int. C. NORTH STETSONAVENUE C07K I4/745 (2006.01) CHICAGO, IL 60601-6731 (US) (52) U.S. Cl. ........................................................ S30/381 (21) Appl. No.: 12/443,428 (57) ABSTRACT The present invention provides conjugates between peptides (22) PCT Fled: Oct. 4, 2007 and PEG moieties through glycerol linkers. Preparation of CMP-SA-Glycerol-PEG-40kDa Yokn---nor-o 3D. oo-()-No,O rA50 mPEG-40 kDa nitrophenyl carbonate CMP-SA-Glycine 1.2g 30.0 g 1)THF/HO (3:1)rt, 5 days HO OH WP O ONC. 2) NaOH, pH 9.5, 1 hr --norO O O &ay 3) TFF, 1 kDa MWCO O HN CO2 Hó 6 4) Q-sepharose anion exchange Yokn- -1o O N HO *N 3. OH 5) TFF, 1 kDa MWCO O nr-450 CMP-SA-Glycerol-PEG-40 kDa 10.11 g, 36% isolated yield 100% purity by UV Patent Application Publication Feb. 18, 2010 Sheet 1 of 47 US 2010/0041872 A1 FIGURE 1 : eqXOy-?Ed-IoueoÁIÐ-VS-dWOjouo??euedeud Patent Application Publication Feb. 18, 2010 Sheet 2 of 47 US 2010/0041872 A1 FIGURE 2 (6z'))"be'Joucºz }IeSuun?poS (60£)"be'Jou! suoppuoouomoeoseqMOv-03d-IoleoÁIÐ-vs-awo %98p???Á6!L’OLp??glund (aujoÁIÐ-VS-dWo"sauuuyzzAn)Aq?und Patent Application Publication Feb. 18, 2010 Sheet 3 of 47 US 2010/0041872 A1 FIGURE 3 sseoouduo?eo???undeqXOV-9Bd-IoueoÁIÐ-VS-dWO ?eseo(TEHIJ º1, (speeg615) ?sOue?d?S-O Á?deu6O?euol?O Patent Application Publication Feb. 18, 2010 Sheet 4 of 47 US 2010/0041872 A1 FIGURE 4 009 s SQL eQX07-9Bd-IoueOÁIÐ-VS-dWO?ouo?eo??undesoue?des-o '(zuL'OAedKLueelos?euelqeueW eso|n||30p??eJeue6ex;eq>||Oy-l)xz) Patent Application Publication Feb. 18, 2010 Sheets of 47 US 2010/0041872 A1 FIGURE 5 o Tri -1 N T --9. gPage6E E GvR.' r SSP's 938649eA. o of 1 site.BESE-R. S w O s'. wr B7778 E s 999 it (D OSSEF.6'sEaseeviv- N t; (6:1 ? E7WES987 l? s O mism O) -ris a'a () 0. 2. D CD D a a. C el O) ? fl. SS N. e S -1 O w l c Y v O e s s 2 w D N 4. used w s S.O ver ld Patent Application Publication Feb. 18, 2010 Sheet 6 of 47 US 2010/0041872 A1 FIGURE 6A Protein Organism GenBank I GenPept PDB 3D At 1g08280 Arabidopsis thaliana ACO11438 AAF 18241.1 Q84WOO BTOO4583 AAO42829.1 (9SGD2 NC003070|NP 1723.05.1 At1g08660/F22O13.14 n.d. At3.g48820/T21J1890 (X-2,3-sialyltransferase ST3GA-IV N-2,3-sialyltransferase A585768 St3Ga Y-2,6-sialyltransferase AJ620651 CAFO5850.1 Siato (X-2,8-sialyltransferase SIAT8A OC-2,8-Sialyltransferase CAG27883 Siat8D (x-2,8-sialyltransferase ST8Si- (Siat8C CMP X-2,6- sialyltransferase NM 177517|NP 803483.1 ST6Ga fragment -- ST3Ga- (Siat4B --- sialyltransferase BOS taurus n.d. AJ748842 CAG44451.1 ST3Gal-lil (Siates ST3Gal-VI (Siat10 SEVEEEEEEEEStóGalNAc-V Bos taurus n.d. AJ620949 CAF06586.1 Q9BEG4 Branchiostonna n.d. AF391289 AAM18873.1 Q8T771 floridae polysialyltransferase Cercopithecus 24.99.- AF210729 AAF17105.1 Q9TTO9 (PST) (fragment) aethiops ST8Sia IV polysialyltransferase Cercopithecus 2.499.- AF210318 AAF17104.1 Q9TT10 (STX) (fragment) aethiops ST8Sial ST3Gall (Siat4 Ciona Savignyi n.d. Aj626814 CAF25172.1 ST3Gal Siata C-2,8- AAE28634 Q64690 polysialyltransferase CAA86822.1 ST8Sia IV Gal (1,314-GlcNAc C AAP22942.1 2,3-sialyltransferase St3Gal Gall1,314-GlcNAc cy AAP22943.1 2,3-sialyltransferase St3Galil (fragment (X-2,3-sialyltransferase CAHO4017.1 ST3Gal (Siat4 na. Issa. C-2,3-sialyltransferase Danio rerio ST3Galil (Siat5) a first CAHO4018.1 Patent Application Publication Feb. 18, 2010 Sheet 7 of 47 US 2010/0041872 A1 FIGURE 6B Organism EC# GenBank 1 GenPept 3D (X-2,3-sialyltransferase Danio rerio n.d. AJ626821 CAF25,179.1 ST3Gal (Siates C-2,3-sialyltransferase ST3Gal IV (Siat4C cC-2,3-sialyltransferase ST3Gal W-r (Siat5 CC-2,6-sialyltransferase ST6Gal Siat 1 EE-2,6-sialyltransferase A634.459 CAG2.568.0.1 ST6GalNAc Siat/B CC-2,6-sialyltransferase ST6GalNAc V (Siat7E) fragment DC-2,6-sialyltransferase AJ646883 ICAG26712.1 ST6GalNAc VI (Siat7F) fragment X-2,8-sialyltransferase AJ715535 CAG29374.1 ST8Sia I (Siat 8A) (fragment) X-2,8-sialyltransferase AJ715543 CAG2938.2.1 ST8Sia Il (Siat 8C) fragment cC-2,8-sialyltransferase CAG29384.1 ST8Sia IV (Siat 8D) cC-2,8-sialyltransferase AJ715546 CAG29385.1 ST8SiaV (Siat 8E) O-2,8-sialyltransferase AJ715551 CAG29390.1 ST8Sia VI (Siat 8F) fragment galactosamide (X-2,6- sialyltransferase ll ST6Gall N-glycan CX-2,8- n.d. sialyltransferase - ST3Gal-related (siatér) St3Gal-V st6GalNAc-V X-2,6-sialyltransferase Drosophila (CG4871) ST6Gall melanogaster (X-2,3-sialyltransferase ST3Ga-V Gaius gains ind AJ627.204it; CAF25503.1SAE C-2,3-sialyitransferase ST3Gall NM 205217NP 990548.1 (C-2,3-sialyltransferase Gallus gallus 2.4.99.- AFO35250 AAC 14163.1 O73724 ST3Gal IV (fragment (X-2,3-sialytransferase Gallus gallus AJ585761 ICAE51385.2 ST3GAL (X-2,6-sialyltransferase Gallus gallus w CAFO5852.1 Siat/b AJ620653 X-2,6-sialyltransferase Gallus gallus X75558 CAA53235.1 Q92182 ST60sal NM 205241NP 990572.1 co-2,6-sialyltransferase Gallus gallus AAE68028.1 O921.83 Patent Application Publication Feb. 18, 2010 Sheet 8 of 47 US 2010/0041872 A1 FIGURE 6C Protein Organism EC GenBank I GenPept Swiss Prot PDB 3D ST6CalNAc - AAE68029.1 74946 CAA52902. NM 20524ONP 990571.1 C-2,6-sialyltransferase Gallus gallus 2.4.99.- X77775 AAE68030.1 Q921.84 ST6GalNAc NM 205233CAA54813.1 NP 990564.1 C-2,6-sialyltransferase Gallus gallus n.d. AJ634455 CAG2.5677.1 ST6GalNAc III (SIAT7C) fragment (C-2,6-sialyltransferase Gallus gallus AJ646877 ICAG26706.1 ST6GalNAc V (SIAT7E) fragment X-2,8-sialyltransferase Gallus gallus 24.99.- U73176 AAC28888.1 P79783 (GD3 Synthase) ST8Sia X-2,8-sialyltransferase Gallus gallus AJ 6994.19 (CAG27881.1 SAT8B OC-2,8-sialyltransferase Gallus gallus AJ699420 CAG27882.1 SAT8C (X-2,8-sialyltransferase Gallus gallus AJ699424 CAG27886.1 SIAT8F O-2,8-syalyltransferase Gallus gallus AJ704564 CAG28697.1 ST8Six-WSAT8C galactosamide X-2,6- Gallus gallus n.d. A627629 CAF29497.1 sialyltransferase ll ST6Galil Gallus qallus AY515.255 AAS83519.1 polysialyltransferase Gallus gallus ST8Sia IV (X-2,3-sialyltransferase ST3Ga AF059321 L13972 AF155238 AF1861.91 O-2,3-sialyltransferase Q16842 ST3 Ga. OOO654 23768 C-2,3-sialyltransferase BC050380 ST3Galil (SiaT6) AF425851 AF425852 AF425853 AF425854 AAO13862.1 AAO13863.1 AAO13870.1 AAO1387.1.1 Patent Application Publication Feb. 18, 2010 Sheet 10 of 47 US 2010/0041872 A1 FIGURE 6E Protein Organism GenBank I GenPept swapo PDB 3D NM O18414 NP 06.0884.1 F O-2,8- 41680 polysialyltransferase BCO27866 ST8Sia IV BCO53657 AAH53657.1 NM OO5668 NP OO5659.1 X-2,8-sialyltransferase L32867 (GD3 synthase) ST8Sia L43494 BCO46158 AAH46158.1 Q93064 AACR53140.1 AAS75783.1 BAA05391.1 CAA548911 NP 003025.1 (X-2,8-sialyltransferase AAA36613.1 Q92186 ST8Sial B51242.1 Q92470 AAC24458.1 Q92746 BCO69584 AAH69584.1 NM OO6011 NP OO60.02.1 O-2,8-sialyltransferase AFOO4668 AAB87642.1 ST8Sial AFOO3092 AAC 15901.2 NM 015879 NP 056963. cC-2,8-sialyltransferase AAC51727.1 O15466 ENSPOOOOOO20221 (fragment) lactosylceramide X-2,3 24.99.9 AF05026 IAAD 14634.1 Q9UNP4 sialyltransferase AF1194.15 AAF66146.1 O94902 BCO65936 AAH65936.1 (ST3Gal W) AY152815 AAO16866.1 AAP65066 AAP65066.1 AY359105 AAC89463.1 ABO18356 BAA33950.1 CAE893.20.1 N- Homo sapiens BCOO6564 BCOO7802 acetylgalactosaminide BCO 16299 C-2,6-sialyltransferase AY358.672 (ST6GalNAc VI) ABO35173 AKO23900 AU507293 AX880950 N- Homo sapiens AF127142 AAFOO102.1 acetylgalactosaminide BCO36705 AAH36705.1 C-2,6-sialyltransferase AAP63349. IV (ST6GalNAc IV) AB035172 BAA87034.1 BAA91281.1 CAB44354.1 CACO7404.1 CAC24981.1 CAC27250.1 CAF 14360.1 NM_014403 NP 055218.3 NM 175039 NP 7782O4.1 ST8SIA-V1 (fragment) Aj621.583 CAF21722.1 homosen a M 291725 XP 291725.2 unnamed protein Homo sapiens AKO21929 BAB13940.1 Q9HAA9 Oroduct AX881696 CAE91353.1 Gali-1,314-GlcNAc C Mesocricetus 2.499.6 A.J245699 CAB53394.1 O90XF6 Patent Application Publication Feb. 18, 2010 Sheet 11 of 47 US 2010/0041872 A1 FIGURE 6F 1 3D auratus MeSOCricetus auratus pope CAB53395.1 Q9QXF5 MeSOCricetus a Fiates AAD33879.1 Ch9WUL1 auratuS MeSOCricetus 24.99.- AJ2457.01 CAB53396.1 Q90XF4 auratus X-2,3-Sialyltransferase Mus musculus 24.994 AF214028 AAF60973.1 P54751 ST3Gal BAC27356.1 Q11202 AKO78469 BAC37.290.1 73523 CAA51919.1 NM OO9177NP 033203.1 cC-2,3-sialyltransferase St3gal2 MuS nuSCulus AAH15264.1 Q11204 ST3Gal AAH66064.1 BAC28752.1 BAC28859.1 C-2,3-sialyltransferase St3gal3 MuS musculus ST3Gail s 3-Sialyltransferase ST3Gal V (X-2,3-sialyltransferase MuS musculus ST3Gal VI ABO63326 BAB79494.1 AKO33562 BAC2836O1 X-2,6-sialyltransferase Stögalnac2 Mus musculus - P70277 ST6GalNAc BCO10208 AAH102081 Q9DC24 AB027198 BABOO637.1 Q9JJM5 AKOO4613 BAB23410.1 X93999 CAA63821.1 X94000 CAA63822.1 NM OO918ONP 033206.2 or-2,6-sialyltransferase Stögal1 MuS musculus 2.499.1 AAE680311 Q64685 ST6Gall BCO27833 AAH27833.1 Q8BM62 D16106 BAA03680.1 O8K1 L1 AKO34768 BAC288281 AKO84124 BAC39120.1 NM 145933NP 666045.1 CE-2,6-sialyltransferase Stögal2 MuS musculus AKO82566 BAC38534.1 O8BUU4 ST6Gal ABO95093 BAC87752.1 AK1294-62 BAC9827.2.1 NM 172829NP 766417.1 ox-2,6-sialyltransferase Stögalnac1 Mus musculus 24.99.3 Y11274 CAA72137.1 Q9QZ39 NMO 11371 INP 0355011 Q9JJP5 ST6GalNAc (X-2,6-sialyltransferase Stögalnac3 Mus musculus ST6GalNAc Patent Application Publication Feb.