US 2015.0017246A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0017246 A1 Huang (43) Pub. Date: Jan. 15, 2015

(54) NOVEL CROSSLINKING REAGENTS, Publication Classification MACROMOLECULES, THERAPEUTIC BIOCONJUGATES, AND SYNTHETIC (51) Int. Cl. METHODS THEREOF A647/48 (2006.01) CI2N II/06 (2006.01) (71) Applicant: CellMosaic, Inc., Worcester, MA (US) (52) U.S. Cl. CPC ...... A61K 47/4823 (2013.01): CI2N II/06 (72) Inventor: Yumei Huang, Lexington, MA (US) (2013.01); A61K 47/48369 (2013.01); A61 K 47/48238 (2013.01) USPC ...... 424/490; 536/1.11: 435/181: 530/391.9; (73) Assignee: CellMosaic, Inc., Worcester, MA (US) 530/402:536/6.4 (57) ABSTRACT (21) Appl. No.: 14/156.224 The invention provides novel chemical entities based on Sugar alcohols. These new chemical entities are biocompat ible and biodegradable. The molecules can be made in a (22) Filed: Jan. 15, 2014 single and pure form. The molecular weights of these mol ecules range from small (<1000 Da) to large (1000-120,000 Da). The Sugar alcohol-based molecules can have functional Related U.S. Application Data groups throughout the molecule for crosslinking compounds, Continuation-in-part of application No. PCT/US2012/ Such as the preparation of antibody-drug conjugates, or to (63) facilitate the delivery of therapeutic proteins, peptides, 047255, filed on Jul. 18, 2012. siRNA, and chemotherapeutic drugs. Also provided are new (60) Provisional application No. 61/509,296, filed on Jul. conjugate entities prepared through Sugar alcohol molecules. 19, 2011, provisional application No. 61/754,571, Methods of synthesizing Sugar alcohol-based molecules and filed on Jan. 19, 2013. conjugates are also within the scope of the invention. Patent Application Publication Jan. 15, 2015 Sheet 1 of 12 US 201S/001 7246 A1

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Patent Application Publication Jan. 15, 2015 Sheet 12 of 12 US 201S/001 7246 A1

US 2015/001 7246 A1 Jan. 15, 2015

NOVEL CROSSLINKING REAGENTS, 0006 wherein MACROMOLECULES, THERAPEUTIC 0007 each M is independently selected from the group BIOCONJUGATES, AND SYNTHETIC consisting of a protein, an enzyme, an antibody, an antibody METHODS THEREOF fragment, a polypeptide, an oligonucleotide, an oligonucle otide analog, a polysaccharide; FIELD OF THE INVENTION 0008 each B is a single MW modified sugar alcohol poly mer, comprising: 0001. The invention relates to novel sugar alcohol-based 0009 from 2 to about 2000 sugar alcohol monomer(s): crosslinking reagents, macromolecules, therapeutic conju 0010 wherein each monomer has from 3 to about 14 gates, and synthetic methods. More specifically, the invention —OR' groups; relates to novel chemical entities that may be used for label I0011 wherein R' is independently selected from ing, conjugation, modification, molecule immobilization, the group consisting of hydrogen, C-C alkyl, ben therapeutic or diagnostic agents, and in drug delivery. Fur Zoyl, acetyl, benzyl, C-C alkylsilyl cyclic ortho thermore, the invention relates to novel conjugates prepared ester, actinide, acetate, tetrahydropyranyl, tetrahy through new Sugar alcohol-based crosslinking reagents and drofuranyl, and tetrahydrothiofurany; macromolecules. 0012 each L is independently selected from the group consisting of: BACKGROUND (0013 a Rand, a structure of V. R V , wherein: 0002 Bioconjugation technologies have been adapted in 0014 V and V are independently selected from the the biotechnology and pharmaceutical industries for the group consisting of: preparation of drug entities, detection reagents, and formula 00.15 Diels-Alder adduct, a 1,3-dipolar tion strategies (Greg T. Hermanson “Bioconjugate Tech (0016 adduct, —C(=G)-G-, -G-C(=G)-, -G-, niques”, 2008, Elsevier Inc.; Christof M. Niemeyer “Biocon G'-C(=G)-G'-, - S S -, - S -(CH2), S(O) jugation Protocols: Strategies and Methods”, 2004, Humana 2 . —S(O) -(CH2), S -, -S(O), N(R)—, Press, Inc.). Bioconjugation is a chemical process that links N(R) S(O) , C(O) NH NH-CH , together two or more biomolecules, including conjugation, C(O) NH N=CH , CH N. NH C labeling, modification, or immobilization of the biomol ecules.

SUMMARY OF THE INVENTION 0003. The present invention provides a novel class of mol ecules based on Sugar alcohol (SA), their applications in conjugate preparation, and methods for synthesizing these SA-based molecules. The invention is based, in part, on SAS with better hydrophilicity and higher hydrodynamic volume than polyethyleneglycol (PEG). The invention is also based, in part, on SAS that can be derivatized and modified to allow for the incorporation of various functional groups and activa tion groups to produce SA crosslinking reagents. The inven tion is also based, in part, on SAS that can be further reacted with various other SAS to prepare single and pure high molecular weight (MW) compounds (SA macromolecules), including, for example, linear SA macromolecules and 3/ Sophisticated two- or three-dimensional architectures, such as branched, cyclic, and hyperbranched SA macromolecules. 0004. The advantages of SA macromolecule compounds of the invention compared to existing compounds include, for example, (i) super hydrophilicity conferred from SAS that can be used to modulate biomolecule properties after conjuga h's tion; (ii) the ability to make crosslinking reagents with ver satile functional groups; (iii) the availability of single and pure higher MW compounds; (iv) multivalent conjugation sites allowing higher loading of drug molecules; (V) an improved hydrodynamic radius; (vi) biodegradablity and bio , the compatibility; and (vii) natural, inexpensive starting materi als. 0017 wherein: 0005. In one aspect, the invention generally relates to a 0018 each G' is independently selected from NR, O, conjugate having a chemical structure selected from the and S; group consisting of 0.019 each G' is independently O or S: I0020 each G is independently selected from S, O, M-(L-B). Formula (I), and NR, and SO; B-(L-M), Formula (II) 0021 each G' is independently O or NR; US 2015/001 7246 A1 Jan. 15, 2015

(0022 each R is independently selected from a bond, -continued C-C alkyl, -(CH2CH2O)o , —(CH2CH2O). 10—CH2—, optionally Substituted alicyclyl, heteroali cyclyl, aryl, a peptide, and a peptidomimetic oligomer, I0023 each Risindependently selected from hydrogen, C-Cs alkyl, —(OCH2CH2), optionally substituted alicyclyl, and optionally substituted heteroalicyclyl; (0024) each R is independently C-Cs alkyl: he 0025 u is an integer from 1 to about 20; and, 0026. In another aspect, the invention generally relates to a conjugate A conjugate having chemical structural Formula (III): , the (M2-L)-B (III) 0027 wherein 0037 wherein: 0028 each M is independently selected from the group 0038 each G' is independently selected from NR, O, consisting of a metabolite, a fluorescent compound, a chemi and S; luminescent compound, a mass tag, a chromophore, biotin, a 0.039 each G' is independently O or S: toxin, a drug, a chemotherapeutic agent, a cytotoxic agent, an I0040 each G is independently selected from S, O, immunosuppressive agent, a diagnostic agent, a radioligand. NR, and SO: and a small molecule, 0041 each G' is independently O or NR; 0029 each B is a single MW modified sugar alcohol poly 0042 each R is independently selected from a bond, mer, comprising: C-C alkyl, -(CH2CH2O)o , —(CH2CH2O). 0.030 from 2 to about 2000 sugar alcohol monomer(s): lo—CH2—, optionally substituted alicyclyl, heteroali 0031 wherein each monomer has from 3 to about 14 cyclyl, aryl, a peptide, and a peptidomimetic oligomer; —OR' groups; I0043) each Risindependently selected from hydrogen, I0032 wherein R is independently selected from C-C alkyl, —(OCHCH), optionally substituted the group consisting of hydrogen, C-C alkyl, ben alicyclyl, and optionally substituted heteroalicyclyl; Zoyl, acetyl, benzyl, C-C alkylsilyl cyclic ortho 0044) each R is independently C-Cs alkyl; ester, actinide, acetate, tetrahydropyranyl, tetrahy 0.045 q is an integer from 1 to about 100. drofuranyl, and tetrahydrothiofurany; 0046. In yet another aspect, the invention generally relates 0033 each L is independently selected from the group a conjugate having a chemical structure selected from the consisting of: group consisting of: 0034) a Rand, a structure of V. R V , wherein: (M1)-L-(B-(L-M2)), (IV) and 0035 V and V are independently selected from the group consisting of: (M-L)-(B-(L-M2)), (V) and 0036 Diels-Alder adduct, a 1,3-dipolar adduct, M1-(L-B-(L-M-2)), (VI) —C(=G)-G-, -G-C(=G')-, -G-, -G-C(=G')-G-, 0047 wherein 0048 each M is independently selected from the group consisting of a protein, an enzyme, an antibody, an antibody fragment, a polypeptide, avidin, Streptavidin, an oligonucle otide, an oligonucleotide analog, a polysaccharide; 0049 each M is independently selected from the group consisting of a metabolite, a fluorescent compound, a chemi luminescent compound, a mass tag, a chromophore, biotin, a toxin, a drug, a chemotherapeutic agent, a cytotoxic agent, an immunosuppressive agent, a diagnostic agent, a radioligand. a small molecule, and another biologically active molecule: 0050 each B is a single MW modified sugar alcohol poly mer, comprising: 0051 from 2 to about 2000 sugar alcohol monomer(s): 0.052 wherein each monomer has from 3 to about 14 —OR' groups; 0053 wherein R' is independently selected from the group consisting of hydrogen, C-C alkyl, ben Zoyl, acetyl, benzyl, C-C alkylsilyl cyclic ortho 3-7. ester, actinide, acetate, tetrahydropyranyl, tetrahy drofuranyl, and tetrahydrothiofurany; US 2015/001 7246 A1 Jan. 15, 2015 3

0054 each L is independently selected from the group S-(L-B-(L-M2)), (DX) consisting of: 0055 a Rand, a structure of V-R V , wherein: S-(L-B-L-(M2)), (X) 0056 V V. re independently selected from the 0070 wherein group cons1Sung OI: 0057 Diels-Alder adduct, a 1,3-dipolar adduct, 0071 S comprises a solid support; —C(=G)-G-, -G-C(=G')-, -G-, -G-C(=G')-G-, 0072 each M is independently selected from the group S—S—, S-(CH2). S(O) , S(O), consisting of a protein, an enzyme, an antibody, an antibody (CH), S , S(O), N(R)— —N(R) S(O) fragment, a polypeptide, avidin, Streptavidin, an oligonucle , —C(O) NH NH-CH , —C(O) NH otide, an oligonucleotide analog, a polysaccharide; N–CH , CH=N. NH C(O) , CH, 0073 each M is independently selected from the group NH NH CO) , N(R) S(O), N(R) , consisting of a metabolite, a fluorescent compound, a chemi —C(O) NH-CH(CHSH) , - N=CH-, luminescent compound, a mass tag, a chromophore, biotin, a NH-CH , NH-C(O)-CH C(O) toxin, a drug, a chemotherapeutic agent, a cytotoxic agent, an NH-, -CH=N-G-, -CH NH-G-, -G -NH immunosuppressive agent, a diagnostic agent, a radioligand. CH-, -G-N=CH-, -C(=NH"). NH-, a small molecule, and another biologically active molecule: NH C NH) , - O P(=O)(O) NH 0074 each B is a modified sugar alcohol polymer, com -NH P(=O)(O)-O-, -CH-CH(NH)– prising: C; (O) to , ", ES5's "5 0075 from 2 to about 2000 sugar alcohol monomer(s): - O P(=S)(S)-O , 0076 wherein each sugar alcohol monomer has from 3 to about 14 —OR' groups: O O 0077 wherein R' is independently selected from X-r the group consisting of hydrogen, C-C alkyl, ben NH N S s Zoyl, acetyl, benzyl, C-C alkylsilyl cyclic ortho ester, actinide, acetate, tetrahydropyranyl, tetrahy O drofuranyl, and tetrahydrothiofurany; O O 0078 wherein each L is independently selected from S -R the group consisting of a R and —V-R° V , N SK 0079 wherein: HN s 0080 V and V are independently selected from the O group consisting of O O 0081 Diels-Alder adduct, a 1,3-dipolar adduct, S , and S —C(=G)-G-, -G-C(=G')-, -G-, -G-C(=G')- N N s G'-, -S-S-, -S-(CH2), S(O). , —S(O) (CH), S , S(O), N(R)—, N(R) O O NH N–CH , CH=N. NH C(O) , - . CH-NH NH-C(O) , N(R) S(O), 1958 wherein N(R) , - C(O) NH-CH(CH2SH) , 0059 each G' is independently selected from NH, O, N -CH s NH CH2 s NH C(O) and S; 2 : - . CH-C(O) NH-, -CH=N-G-, -CH 0060 each G s independently O or S: NH-G-, -G-NH-CH , -G-N=CH , 0061 each G is independently selected from S. O. —C(=NH) NH-, -NH C(=NH)– NR,3. and SO; - O P(=O)(O) NHs , NH P(=O)s 0062 each G' is independently O or NR; (O)—O , CH-CH(NH)—CH2—S , 0063 each R is independently selected from a bond, S CH-CH(NH)—CH2—, O P(=O) C-C alkyl, -(CH2CH2O). , —(CH2CH2O). (O)-O-, - O P(=O)(S)-O-, -O-P 10—CH2—, optionally Substituted alicyclyl, heteroali- (—S)(S)-O , cyclyl, aryl, a peptide, and a peptidomimetic oligomer, I0064 each Risindependently selected from hydrogen, C-Cs alkyl, —(OCH2CH2), optionally substituted O O alicyclyl, and optionally substituted heteroalicyclyl; y R I0065 each R is independently C-Cs alkyl: NN S s 0.066 u is an integer from 1 to about 100; NH 0067 q is an integer from 1 to about 100; and, O 0068 k is 0 or an integer from 1 to about 20. O 0069. In yet another aspect, the invention generally relates S R O to a conjugate having chemical selected from the group con- N1 S{ sisting of: HN s S-(L-B-(L-M1)), (VII) O US 2015/001 7246 A1 Jan. 15, 2015

-continued —O N-(Boc) —O N(-phthalimidyl), —NH NH, C(=O) NH NH, NH CO-O) NH-NH, -NH C(=S) NH NH, -toluene sulfonylhydrazide, -R NH-C(=NH) NH, a benzophenone, an aryl diazonium, a diaZoalkane, a diazoacetyl, an anthraquinone, a diazirine, an option ally substituted trifluoromethylphenyldiazirine, a , the diene, a dienophil, a 1,3-dipole, a dipolarophile, an 0082 wherein: alkene, a ketene, an olefin, an alkene with allylic I0083 each G' is independently selected from NH, O, hydrogen, a dicarbonyl group, an epoxide, an oxirane, and S; an organosilane, a phosphonium group, an ester, an I0084 each G’ is independently O or S: anhydride, a carbonate group, a glyoxal, I0085 each G is independently selected from S, O, —C(=NH)—O R, a hydroxymethyl phosphine NR, and SO: derivative, an ethyl vinyl, a maleimide, a vinylsul I0086) each G' is independently O or NR; fone, an allyl Sulfone, a thioester, a cisplatin deriva I0087 each R is independently selected from a bond, tive, an aziridine, an acryloyl group; C-C alkyl, -(CH2CH2O). , —(CH2CH2O). 10100 each R is independently selected from hydro 10—CH2—, optionally Substituted alicyclyl, heteroali gen, C-C alkyl, alicyclyl, heteroalicyclyl, benzyl or cyclyl, aryl, a peptide, and a peptidomimetic oligomer, aryl, wherein any ring in R is optionally substituted; I0088 each Risindependently selected from hydrogen, 10101 each R is independently selected from ben C-Cs alkyl, —(OCH2CH2), optionally substituted Zoyl, acetyl, benzyl, C-C alkylsilyl, tetrahydropy alicyclyl, and optionally substituted heteroalicyclyl; ranyl, tetrahydrofuranyl, and tetrahydrothiofuranyl: I0089 each R is independently C-Cs alkyl: 0090 u is an integer from 1 to about 500; and, 10102) each R" is independently selected from trityl, 0091 k is 0 or an integer from 1 to about 20. MMT, and DMT: 0092. In yet in another aspect, the invention is generally (0103) each R is independently selected from 2-py relates to a single MW compound having a linear, branched or ridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, macrocyclic multimer Sugar alcohol comprising three or 2-nitrophenyl, 4-nitrophenyl, 3-carboxy-4-nitrophe more monomeric Sugar alcohol unit B'; and nyl, and 2,4-dinitrophenyl: 0093 each monomeric sugar alcohol unit is bound to one or more other monomeric units through a linking 0104 each J is independently selected from Cl, Br group W formed by a reaction between the X, Y or Z. and I portion of one monomeric unit with the X, Y or Z of 0105 each of L', L, and L is independently a Ror another monomeric unit; -R V R *, wherein: 0094 wherein 0106 “*” represents a portion of L', L, and Li 0095 each B" has the chemical structural Formula XV: bound to X, Y. S. M or M, or a Z, respectively; 0107 each V and Ware independently selected from

(XV) the group consisting of Diels-Alder adduct, a 1.3- dipolar adduct, —C(=G)-G'-, -G-C(=G)-, -G-, -G-C(=G)-G'-, - S S , —S (CH), S(O) , —S(O) (CH), S -, -S(O), N(R)—, N(R) S(O) , C(O) NH NH-CH , C(O) NH N=CH , CH N. NH C (O) , —CH-NH-NH C(O) , N(R)-S (O). N(R)-, -C(O) NH-CH(CHSH) , N–CH , NH-CH , NH C(O) 0096 wherein for each B", independently: 0097 each of n and p is independently selected from 0 and an integer selected from 1 to about 12; and n+p is between 1 and 12; 0.098 r is 0 or 1; (0099 each of X, Y and Z is independently selected from -OH, -J., RJ, —C(=O)-J, —C(=O)— CH-J, NH C(=O) CH-J, OR, OR, —OR. -O-Mesyl, -O-Tosyl, -NH C(=O)— CH-O-Mesyl, - NH C(=O)—CH O-Tosyl, -SH, -S-S-tButyl, - SR7, -SR, -S-S R. –S(=O).-J., NH, -NHR, N(R)R, -NHR'. - NH-Fmoc, -NH-Boc, -C(=O)H, —C(=O) R, C(=O)CH, N–C–S, N–C–O, - C=C R, N—N*—N, 3-7. —O NH, —O NH-Fmoc. —O NH-Boc, US 2015/001 7246 A1 Jan. 15, 2015

-continued Boc, —O N-(Boc) —O N(-phthalimidyl), —C(=O)— NH NH, a phenol group, an optionally substituted trifluo romethylphenyldiazirine, a diene, a dienophil, a 1,3-dipole, a dipolarophile, an alkene, a ketene, an olefin, an alkene with allylic hydrogen, an optionally Substituted N-hydroxysuccin imide ester, a imidoester, a maleimide, a phosphoramidite; I0124 each ofY is independently selected from —S S h's tEutyl, - SR", -S-S-R, -NH C(=O)-CHCH S–SR, NHR'. - NH-Fmoc, -NH-Boc, O NH, —O NH-Fmoc, —O NH-Boc, —O N-(Boc) —O N (-phthalimidyl), an optionally substituted trifluorometh ylphenyldiazirine, an optionally substituted N-hydroxysuc cinimide ester, a imidoester, a maleimide; , the I0125 each R" is independently selected from the group consisting of hydrogen, C-C alkyl, benzoyl, acetyl, benzyl, 0.108 wherein: C-C alkylsilyl cyclic ortho ester, actinide, acetate, tetrahy 0109) each G' is independently selected from NH, dropyranyl, tetrahydrofuranyl, and tetrahydrothiofurany; O, and S; 0110 each G’ is independently O or S; I0126) each R is independently selected from benzoyl, I0111 each G is independently selected from S.O. acetyl, benzyl, C-Cs alkylsilyl, tetrahydropyranyl, tetrahy NR, and SO: drofuranyl, and tetrahydrothiofuranyl: 0112 each G' is independently O or NR; (O127 each R7 is independently selected from trityl, MMT, I0113) each R is independently selected from a and DMT: bond, C-C alkyl, -(CH2CH2O), . I0128 each R is independently selected from 2-pyridyl, —(CH2CH2O)o CH-13 , optionally substi 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophe tuted alicyclyl, heteroalicyclyl, aryl, a peptide, and nyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, and 2,4-dinitro a peptidomimetic oligomer, phenyl: I0114) each R is independently selected from I0129 each J is independently selected from Cl, Br, and I hydrogen, C-C alkyl, -(OCH2CH), option I013.0 L and L is independently selected from a bond, ally substituted alicyclyl, and optionally substi CH, *, —C(=O) NH-Cls *, —CH NH tuted heteroalicyclyl: ce-O) C1-s aikvi 3, CH C(=O) NH C1-8 aikvi 0115 each R is independently C-Cs alkyl: I0116 each R is a bond or —CH2—, 0131 “*” represents a portion of Land Li bound X" or 0.117 and Yi; I0118 at least in one of the B' unit each -L-Z 0.132. In yet another aspect, the invention is generally portion is —OR'; wherein each R" is indepen relates to a dimeric Sugar alcohol having the chemical struc dently selected from the group consisting of hydro tural Formula XVIII: gen, C-C alkyl, benzoyl, acetyl, benzyl, C-Cs alkylsilyl cyclic ortho ester, actinide, acetate, tet rahydropyranyl, tetrahydrofuranyl, and tetrahy (XXVIII) drothiofurany; I0119 at least in one of the B' unit, n+p+r is greater than 1. 0120 In yet another aspect, the invention is generally relates to a monomeric Sugar alcohol having the chemical structural Formula XXVI: 0133) each of n1, m2, p 1, and p2 is independently selected from 0 and an integer selected from 1 to about (XXVI) 12; and n1+p2 is between 1 and 12, n2+p2 is between 2 and 12; 0.134 r is 0 or 1: 0135) wherein W is selected 0.136 from —S , —O , NH , NC-Calkyl-, - C(=O)NH-, -NHC(=O) , S(=O) , —S(=O), —P(=S),O , and - P(=S)(=O)C . 0121 each of n and p is independently selected from 0 and 0.137 each of X is independently selected from -OH, an integer selected from 1 to about 12; and n+p is between 2 -J, C(=O)-CH-J, -OR. -S-S-R, NH and 12; C(=O). CHCH S S R. S. C(=O)-CH, 0.122 r is 0 or 1: —C(=O)H, C(=O) R.-C(=O)CH, -C=C- (0123 each of X is independently selected from OH, -J. R, N=N*—N, O NH. O. NH-Fmoc, —C(=O)—CH-J, OR, S. S. R. NH C —O NH-Boc, —O N-(Boc) —O N(-phthalim (=O). CHCH-S-S R. —S—C(=O)—CH, idyl), —C(=O)—NH-NH2, a phenol group, an —C(=O)H, -C(=O) R. C(=O)CH, -C=C R, optionally substituted trifluoromethylphenyldiazirine, a - N=N*—N, - O NH, - O NH-Fmoc, O. NH diene, a dienophil, a 1,3-dipole, a dipolarophile, an alk US 2015/001 7246 A1 Jan. 15, 2015

ene, a ketene, an olefin, an alkene with allylic hydrogen, -continued an optionally Substituted N-hydroxySuccinimide ester, a O O imidoester, a maleimide, a phosphoramidite; I0138 each of Y is independently selected from —S—S-tButyl, SR", S S R, NH C (=O). CHCH-S-S-R, -NHR'. - NH-Fmoc, - NH-Boc, O NH, O. NH-Fmoc, O. NH Boc, —O N-(Boc) —O N(-phthalimidyl), an optionally substituted trifluoromethylphenyldiazirine, an optionally Substituted N-hydroxySuccinimide ester, a imidoester, a maleimide; I0139 each R" is independently selected from the group consisting of hydrogen, C-Cs alkyl, benzoyl, acetyl, benzyl, C-C alkyl silyl cyclic ortho ester, actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl, and tet rahydrothiofurany; I0140 each Risindependently selected from hydrogen, C-Cs alkyl, alicyclyl, heteroalicyclyl, benzyl or aryl, wherein any ring in R is optionally substituted; I0141 each R is independently selected from benzoyl, acetyl, benzyl, C-C alkylsilyl, tetrahydropyranyl, tet rahydrofuranyl, and tetrahydrothiofuranyl: O

I0142 each R" is independently selected from trityl, -- MMT, and DMT: CIHN H I0143 each R is independently selected from 2-py N ridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, No r X 2-nitrophenyl, 4-nitrophenyl, 3-carboxy-4-nitrophe O nyl, and 2,4-dinitrophenyl: 0144 each J is independently selected from Cl, Br, and S O I (0145 Land L is independently selected from a bond, R18, W3, —CH2—, —C(=O)—NH Cs aii'? *, —CH2— o1 NH-C(=O)—Cs is -, -CH2 C(=O)— NH-C-si”, O 0146) “*” represents a portion of Land Lbound XorY. NH2, OH 0147 In another aspect, the invention is generally relates to a compound having the structure x H - N=C=S, NECEO and

0148 wherein 0149 n is an integer from 2 to about 8: 0150 m is an integer from 1 to about 8: 0153 each Y is selected from the group consisting of: 0151 p is an integer from about 1 to about 2000; I0152 each of X" is selected from the group consisting O O O of: JI SR18 OR18 O O SR 18, -NR18, or --tor O O O ONH2, --. x - R18 O --coJ| --outSR18 -- OR18 US 2015/001 7246 A1 Jan. 15, 2015

-continued -continued N NO N ON N W1 CF,N > r N

O xO NO2 and C

-- CIHN H N 0158 Each of W is independently selected from the No J| r X group consisting of s O O O E O SONa RI 8, o1W3 s O, O s F F F F O

NH2' ' ' '4' OH x N F F F N=C=S, NECEO and 0159. In one aspect, the invention is generally relates to a Sugar alcohol-derived compound having the structure.

Nuespi I0154 each of W' is selected from the group consisting of C(=O) NH-, -NH C(=O) , (0160 wherein (O155 each of J is selected from the group consisting of 0.161 n is an integer selected from about 2 to about 8: Cl, Brand I; (0162 X is a chemical- or photocrosslinking group I0156) R' is selected from the group consisting of Selected from the groups consisting of: hydrogen, C-st: alicyclyl, heteroalicyclyl, benzylor aryl, wherein any ring in R' is optionally substituted (O157 each of W is independently selected from the group consisting of -- you. --ont. --co --out -- --. --co --on. O WNu y US 2015/001 7246 A1 Jan. 15, 2015

-continued (0166 Each of W’ is independently selected from the N group consisting of M N S CF3, 1. > O

COOH NO O NO NN CIHN H N No s JI ~ X s 2 O NO s s O o x.o1 W3 O

NH2 and OH: NO ON NO x H - (0163 Y is a chemical- or photocrosslinking group xO and selected from the group consisting of: (0167. Each of W is independently selected from the group consisting of O

O O W NuN \ CIHN SONa

O s No s O, O s F F F F

O

W3 ONH o1 2 -R-F and --F F

0.168. In yet another aspect, the invention is generally relates to a compound having the structure. N M N 2 S OH OH WI CF W&Ns/ s and n- ). GL21, W2 S S-N-ry (0169 wherein O s 0170 n is an integer selected from 2 to about 8: 0171 m is an integer selected from 1 to about 8: (0164 W is an independent linker selected from the 0172 p is an integer selected from 2 to about 2000; group consisting of —C(=O)—NH-, and, —NH-C (=O)—: (0165 Each of J is independently selected from Cl, Br and I; US 2015/001 7246 A1 Jan. 15, 2015 9

(0174 each of X andY is independently a chemical- or (0176) L' and L is independently selected from a bond, photocrosslinking group selected from the group con- —CH *, sisting of (0177) “*” represents a portion of L' and Lbound X or Y3. (0178) R' is hydrogen, Cs i? alicyclyl, heteroalicy clyl, benzylor aryl, wherein any ring in R' is optionally SR', -NR18, OR 18, COR18, Substituted. (0179 each of W' is an independent linker selected from the group consisting of —C(=O)—NH —NH C O O O (=O)— s s s 0180 each of J is independently selected from Cl, Br J| SR18 OR18 and I; 0181 each of W is independently selected from the COOH ONH J| group consisting of s 22 s

H W2Ns1 S y W2Ns 1 S N- N X. NO ON NO O CIHN y s r and s No O 0182 each of W is independently selected from the O group consisting of o s -W, R18 O O O SONa N N -NH2, OH N O, O s F F F

N=C=S, NECEO,

NH-NH2: -R-F and F F 0183. Additionally, the present invention provides meth ods for synthesizing SA crosslinking reagents and SA mac 0.175 a diene, a dienophil, a 1,3-dipole, a dipolarophile, romolecules. an alkene, a ketene, an olefin, an alkene with allylic 0.184 The foregoing aspects and embodiments of the hydrogen, a benzophenone, an aryl diazonium, a vinyl- invention may be more fully understood by referencing the Sulfone and an allyl Sulfone; following figures, detailed description, and claims. US 2015/001 7246 A1 Jan. 15, 2015

BRIEF DESCRIPTION OF THE FIGURES oligo conjugates, protein-oligo conjugates, antibody-enzyme conjugates, antibody-protein conjugates, protein-protein 0185 FIG. 1 illustrates the chemical structures, MWs, and conjugates, protein-peptide conjugates, protein-oligo conju Log Pvalues of Sugar alcohol-based linkers and polyethylene gates, fluorescent compounds, immobilized proteins, immo glycol-based linkers. bilized peptides, immobilized enzymes, and immobilized oli 0186 FIG. 2 illustrates the general methods for synthesiz gOS. ing monofunctional, homobifunctional, and heterobifunc (0199 The term “sugar alcohol” (SA) or “sugar alcohols' tional SA crosslinking reagents. (SAS), as used herein, refers to a Sugar alcohol or keto Sugar 0187 FIG.3 illustrates the general methods for synthesiz that has a general formula of OH-CH (CHOH)—(C(O)) ing monofunctional, homobifunctional, and heterobifunc -(CHOH), CH, OH, wherein r is 0 or 1, n and pranges tional SA crosslinking reagents with extra linkers. from 0 to approximately 12 with the combined value of n and 0188 FIG. 4 illustrates examples of useful crosslinking p being greater than 1. When n is O, SA refers to the hydro reagents. genated form of carbohydrate whose carbonyl group (alde 0189 FIG.5 illustrates examples of useful SA reagents for hyde or ketone) has been reduced to a primary or secondary Solid phase peptide and oligo synthesis. OH group. In some cases, SA refers to polyol, polyhydric 0.190 FIG. 6 illustrates methods of synthesizing di-, tetra-, alcohol, or polyalcohol. A Sugar alcohol has the general for and higher MW SA molecules. mula HO—CH2—(CHOH), CH, OH. Examples of (0191 FIG. 7 illustrates the MW of linear SA macromol natural Sugar alcohols are glycol (2-carbon), glycerol (3-car ecules based on D-mannitol. bon), erythritol (4-carbon), threitol (4-carbon), arabitol 0.192 FIG. 8 illustrates the general methods for incorpo (5-carbon), xylitol (5-carbon), ribitol (5-carbon), mannitol rating crosslinking groups at the side chain of SA macromol (6-carbon), sorbitol (6-carbon), dulcitol (6-carbon), and iditol ecules. (6-carbon). Sugar alcohols can also be synthetic. When ris 1, 0193 FIG. 9 illustrates examples of two linear SA macro Sugar alcohol refers to a keto Sugar or ketose. Examples of molecules synthesized by Solid phase. natural ketoses are dihydroxyacetone (3-carbon), erythrulose 0194 FIG. 10 illustrates examples of a few different con (4-carbon), ribulose (5-carbon), xylulose (5-carbon), fructose figurations of hyperbranched SA macromolecules. (6-carbon), psicose (6-carbon), Sorbose (6-carbon), tagatose 0.195 FIG. 11 illustrates examples of SA macromolecules (6-carbon), sedoheptulose (7-carbon). based on D-mannitol. 0200. The term "sugar alcohol unit (SA unit) or “sugar (0196. FIG. 12 illustrates a general method of synthesizing alcohol units” (SA units), as used herein, refers to an branched SA macromolecules. “unmodified’ or “modified mono sugar alcohol. An “unmodified SA unit has the general formula —CH2— DEFINITIONS (CHOH), (C(O)), (CHOH), CH, wherein ris 0 or 1, 0197) The term “conjugation” or “bioconjugation', as nand p ranges from 0 to approximately 12 with the combined used herein, refers to a chemical process that links two or value of n and p being greater than 1. A “modified SA unit more molecules together to create new molecules. One of the refers to a SA unit in which one or more of its OH groups has molecules is preferably a biomolecule. Thus, “bioconjuga been chemically modified by substitution with another func tion” or "conjugation” refers to any chemical process that tional group. A “modified SA unit also refers to a SA unit in involves changing a molecule's properties through covalent which one or both of its —CH2OH groups has been oxidized modification, labeling, conjugation, or immobilization. Con and then further modified with other functional groups. A jugation reactions include, for example, amide bond forma "modified SA unit also refers to a SA unit wherein the tion through pre-activated carboxylate, such as NHS ester hydrogen atom at one or more of the OH groups has been formation with amine; thioether formation through the reac replaced by a chemical protecting group, leaving group, or tion of sulfhydryl with maleimide or alkyl halide; hydrazone other functional group. Examples of OH protecting groups formation through the reaction of hydrazine with ketone or include C-C alkyl, benzoyl, acetyl, benzyl, C-C alkyl aldehyde; oxime formation through the reaction of aminooxy silyl, tetrahydropyranyl, tetrahydrofuranyl, and tetrahy with ketone or aldehyde; semicarbazone formation through drothiofuranyl. the reaction of semicarbazide with ketone or aldehyde; and 0201 The term “monomer refers to a molecule that can reductive amination to conjugate aldehydes and amines. bind chemically to other molecules to form a polymer. The Other less common conjugation reactions include click term "monomeric' as used herein refers to an “unmodified’ chemistry (Cu(I)-promoted azide-alkyne 3+2] cycloaddi or “modified mono sugar alcohol. tion), the Diels-Alder reaction, and photochemical reactions 0202 The term “dimeric', as used herein, refers to a involving azide. chemical entity consisting of two monomeric Sugar alcohols 0198 The term “conjugate’, as used herein, refers to a chemically binding to each other. product produced by a "conjugation” reaction of two or more 0203 The term “trimeric', as used herein, refers to a molecules. Examples of molecules that can be conjugated chemical entity consisting of three of the monomeric Sugar include Small molecules, antibodies and their fragments, pro alcohols. teins (soluble and membrane proteins), enzymes, nucleic 0204. The term “tertrameric', as used herein, refers to a acids and their analogs, peptides and peptidomimetics, fluo chemical entity consisting of four of the monomeric Sugar rescent compounds, chemiluminescent compounds, radioac alcohols. tive compounds, isotopic containing compounds, biotin and 0205 The term “biocompatible’, as used herein, refers to avidin/streptavidin, toxins, drugs, Solid Support media, and the possession of a property by a compound that makes it other biologically active molecules. Examples of conjugates biologically compatible, e.g., by not producing a toxic, inju include antibody-drug conjugates, protein-drug conjugates, rious, or immunological response in living cells, tissues, or a peptide-drug conjugates, oligo-drug conjugates, peptide living system. The term “biocompatibility” as used herein US 2015/001 7246 A1 Jan. 15, 2015 refers to the ability of a biomaterial to initiate an appropriate Organic Chemistry: Reactions, Mechanisms, and Structure, host response in a specific application. In another sense, the 4' Ed., John Wiley and Sons, New York, 1992, 352-357. term “biocompatibility” means the quality of not having toxic or injurious effects on biological systems. In the case of a 0211. The term “protecting group” (PG), as used herein, medical therapy, “biocompatibility” refers to the ability of a refers to a molecular group or chemical moiety that blocks a biomaterial to perform its desired function without eliciting functional group from reacting during other chemical opera any undesired local or systemic effects in the recipient or tions/transformations. A protecting group is inert to these beneficiary of that therapy, as it generates the most appropri chemical operations/transformations but can be removed or ate beneficial cellular or tissue response in that specific situ cleaved by specific chemical, enzymatic, or photochemical ation and optimizes the clinically relevant performance of means in Such a way that it liberates the original functional that therapy. group for further reaction. A wide variety of protecting 0206. The term “biodegradable', as used herein, refers to groups are available and known in the art. An extensive the possession of a property by a compound that allows it to description of protecting groups of typical art can be found in: decompose through one or more in Vivo biological processes, Theodora W. Green and Peter G. M. Wuts, Protective Groups Such as via a metabolic pathway. As used herein, "biodegrad in Organic Synthesis, 2" ed., Wiley-Interscience, New York, able' compounds are those that, when taken up by cells, can 1991. be broken down by the lysosome pathway or other biochemi cal machinery or by hydrolysis into components that the cells 0212. The term “OH protecting group', as used herein, can either reuse or dispose of without significant toxic effects refers to a molecular group or chemical moiety that blocks an on the cells. OH group from reacting during other chemical operations/ 0207. The terms “crosslink', “crosslinking. transformations. Examples of chemical moieties include, but “crosslinked, and grammatical derivatives thereof, refer to are not limited to, alkyl, aryl, benzoyl, acetyl, benzyl, alkyl the covalent bonding or bonds between molecules or between silyl, tetrahydropyranyl, tetrahydrofuranyl, and tetrahy molecules and Solid Supports. drothiofuranyl. 0208. The terms “crosslinking group”, “functional 0213. The terms “linker” or “linkage' or “linking group', group', 'activated group', and “chemically reactive group'. as used herein, refer to groups or bonds that are normally as used herein, refer to distinct, definable portions or units of a molecule that react readily with electrophilic or nucleo formed as the result of a chemical reaction and typically with philic groups on other molecules to form a new molecule covalent bond(s). A linker may contain an extra spacer(s), through covalent bonding. Crosslinking groups include, for such as ethylene glycol, methylene, a peptide, or a peptido example, OH, protected OH, carboxylic acid, protected car mimetic oligomer. Linkers include, for example, Substituted boxylic groups, amines, protected amines, thiols, protected or un-substituted heteroalicyclyl C-C alkyl, thiols, disulfides, alkyl groups, benzophenones, anthraquino —(CH2CH2O)o , —(CH2CH2O). CH2—, Substi nes, diazo groups, azido groups, acyl azides, alkynes, diazo tuted or un-Substituted alicyclyl, heteroalicyclyl, aryl, pep nium groups, diazirenes, dienes, dienophils, 1,3-dipoles, tides, and peptidomimetic oligomers. The linkers may dipolarophiles, alkenes, ketenes, olefins, alkenes with allylic include linking groups, such as acyl-based linking groups hydrogen, dicarbonyl groups, epoxides, oxiranes, organosi (e.g., —C(O)—NH and—OC(O)NH ). Exemplary link lanes, isothiocyanate, isocyanate, phosphonium groups, tosy ing groups include, but are not limited to, each V and W lates, mesylates, acyl azides, esters (e.g., N-hydroxysuccin independently selected from a Diels-Alder adduct, a 1,3- imidyl ester and 1-benzotriazolyl esters), sulfonyl chlorides, dipolar adduct, —C(=G)-G-, -G-C(=G)-, -G-, -G-C anhydrides, tetrahydropyranyl groups, tetrahydrofuranyl (G)-G'-, -S-S-, -S-(CH2). S(O). , —S(O) - groups, tetrahydrothiofuranyl groups, carbonate groups (e.g., (CH), S , S(O), N(R)-, - N(R) S(O) , N-hydroxysuccinimidylcarbonates and 1-benzotriazolyl car C(O) NH-NH-CH , C(O) NH N=CH-, bonates), aldehydes, ketones, aryl ketones, glyoxals, imi CH=N. NH C(O) , CH NH NH C(O) , doesters, anhydrides, fluorophenyl esters, hydroxymethyl phosphine derivatives, haloacetyl groups, ethyl vinyls, aryl - N(R) S(O). N(R) , C(O) NH CH halides, trityl halides, alkylhalides, acylhalides, silyl halides, (CHSH) ,-N=CH-, -NH-CH , NH-C(O) maleimides, vinylsulfones, thioesters, cisplatin derivatives, CH-C(O) NH-, -CH=N-G-, -CH-NH-G-, fluorobenzene derivatives, aziridines, acryloyl groups, ami -G-NH-CH , -G-N=CH C(-NH) NH , nooxy, protected aminooxy, semicarbazide, thiosemicarba NH-C(=NH) , - O P(=O)(O) NH Zide, hydrazine, guanidinyl, phosphoramidites, and Sugar - NH P(=O)(O)-O-, -CH-CH(NH) CH groups. An extensive description of such groups of typical art S-, -S CH-CH(NH)-CH - O P(=O)(O)– can be found in the following reference: Greg T. Hermanson O - O P(=O)(S)-O-, - O P(=S)(S)-O-, “Bioconjugate Techniques”, 2008 Elsevier, Inc. 0209. The term “crosslinking reagent, as used herein, refers to a molecule that includes a crosslinking group and is capable of crosslinking with another molecule. 0210. The term “leaving group', as used herein, refers to a chemical moiety that can be substituted with another chemi cal moiety. Examples of leaving groups include halides (e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-tolu h' 3-1 enesulfonyl (tosyl), trifluoromethyl sulfonyl (triflate), and trifluoromethylsulfonate. An extensive description of leaving groups of typical art can be found in: Jerry March, Advanced US 2015/001 7246 A1 Jan. 15, 2015 12

-continued amino acids. The term peptide may also apply to amino acid polymers in which one or more amino acid residues are arti ficial chemical analogs of a corresponding naturally occur ringamino acid. An amino acid polymer in which one or more amino acid residues is an "unnatural amino acid not corre sponding to any naturally occurring amino acid is also encompassed by the term "peptide'. 0220. The term “protein', as used herein, refers to a poly mer of amino acid residues linked together by a peptide bond. The term is meant to include proteins and polypeptides of any size, structure, or function. However, a protein is typically at least 10 amino acids long. A protein may be naturally occur ring, recombinant, synthetic, or any combination of these. A protein may also be a fragment of a naturally occurring pro , the tein. A protein may be a single molecule or it may be a multi-molecular complex. The term protein may also apply to wherein each G' is independently selected from NR. O. and amino acid polymers in which one or more amino acid resi S; each G’ is independently O or S; each G is independently dues are artificial chemical analogs of a corresponding natu selected from S. O. NR, and SO; each G' is independently rally occurring amino acid. An amino acid polymer in which O or NR; each R is independently selected from hydrogen, one or more amino acid residues is an "unnatural amino acid C-C alkyl, -(OCH2CH), optionally substituted alicy not corresponding to any naturally occurring amino acid is clyl, and optionally substituted heteroalicyclyl; each R is also encompassed by the use of the term “protein'. independently C-C alkyl; 0221) The term “protein fragment, as used herein, refers 0214. The term “alkyl, as used herein, refers to a to a peptide that is a portion of another protein. For example, branched, unbranched, or cyclic hydrocarbon having, for protein fragments may be polypeptides obtained by digesting example, from 1 to 20 carbon atoms, and often 1 to about 12, a full-length protein. A protein fragment typically comprises 1 to 6, or 1 to 4 carbon atoms. Examples include, but are not at least two amino acids. limited to, methyl, ethyl, 1-propyl. 2-propyl, 1-butyl, 2-me 0222. The term “therapeutic agent, as used herein, refers thyl-1-propyl, 2-butyl, 2-methyl-2-propyl (t-butyl), 1-pentyl, to a compound, or a molecule that is useful in the treatment of 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, a disease. Therapeutic agents include, for example, antibody 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, fragments, drugs, toxins, nucleases, hormones, immuno 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2- modulators, pro-apoptotic agents, anti-angiogenic agents, pentyl, 3-methyl-3-pentyl, 2-methyl-3-penty1, 2,3-dimethyl boron compounds, photoactive agents, dyes and radioiso 2-butyl, 3.3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl topes, proteins, and constructs that include proteins, oligo and the like. The alkyl can be unsubstituted or substituted. nucleotides, oligonucleotide analogs, polysaccharides, The alkyl can also be optionally partially or fully unsaturated. metabolites, enzymes, polypeptides, and toxins. Therapeutic As such, the recitation of an alkyl group includes both alkenyl agents include prodrugs of bioactive agents and constructs. and alkynyl groups. 0223) The term “therapeutic moiety', as used herein, 0215. The term “SA molecule', as used herein, refers to a refers to a functional moiety that is derived from a “therapeu molecule that includes an SA unit. SA molecule refers to any tic agent'. of the SA crosslinking reagents, SA macromolecules, linear 0224. The term “diagnostic agent’, as used herein, refers SA macromolecules, branched SA macromolecules, and to a compound, or a molecule that alone or in combination hyperbranched SA macromolecules. with another agent is able to be used for revealing, pinpoint 0216. The term “SA macromolecule' as used herein refers ing, and defining the localization of a pathological process. to a high molecular weight compound derivatized from the Diagnostic agents include, for example, radioactive Sub Sugar alcohol. Typically, an SA macromolecule is at least two stances, fluorescent dyes, chemiluminescent compounds, SA units long. The preferred size of an SA macromolecule mass tags, chromophores, biotin, toxins, proteins, enzymes, ranges from approximately 1000 Da to approximately 120, antibodies, antibody fragments, polypeptides, avidin, Strepta OOODa. vidin, oligonucleotides, oligonucleotide analogs, polysac 0217. The term “branching, as used herein, refers to the charides, metabolites, drugs, chemotherapeutic agents, cyto replacement of a Substituent, for example a hydrogenatom on toxic agents, immunosuppressive agents, and radioligands. a Sugar alcohol, by another covalently bonded chain of a Sugar 0225. The term “diagnostic moiety', as used herein, refers alcohol, or by a chain of another type. to a functional moiety that is derived from a "diagnostic 0218. The terms “macrocycle' or “macrocyclic”, as used agent'. herein, refer to a cyclic macromolecule or a macromolecular 0226. The term “a biologically functional moiety', as used cyclic portion of a molecule. Typically, a macrocycle includes herein, refers to a moiety that can elicit some kind of biologi a 7- or greater membered ring. cal function or interact with biological systems to elicit some 0219. The terms “peptide' or “polypeptide', as used kind of biological function. “Biologically functional moiety’ herein, refer to a polymer of amino acid residues linked also refers to a moiety that can aid in detecting or diagnosing together by a peptide bond. Typically, a peptide is at least two Some biological function. Examples of biologically func amino acids long. A peptide bond is commonly known in tional moieties include therapeutic moieties and diagnostic biochemistry as an amide linkage between the carboxyl group moieties. of one amino acid and the amino group of anotheramino acid. 0227. The term “antibody', as used herein, refers to a The preferred size of peptides ranges from about 2 to about 40 full-length immunoglobulin molecule oran immunologically US 2015/001 7246 A1 Jan. 15, 2015 active portion of an immunoglobulin molecule. Such as an two or more glycerol units. Polyglycerol is prepared by gen antibody fragment. Antibody' is used in the broadest sense eral polymerization of glycerol at a higher temperature or and includes monoclonal antibodies, polyclonal antibodies, under basic conditions. multispecific antibodies (e.g., bispecific antibodies), and 0232. The term “hydrophobic moiety', as used herein, antibody fragments. The immunoglobulin disclosed herein refers to a nonpolar molecule or group that has little affinity can be of any type, for example, IgM, Ig|D, IgG, IgE, IgA, or for water. A hydrophobic molecule orportion of a molecule is any Subclass of immunoglobulin, Such as IgG1, IgG2a, one that has a tendency to cluster together with other hydro IgG2b, IgG3, IgA1, and IgA2. Antibodies may be murine, phobic groups in an aqueous environment because they are unable to disrupt the network of strong hydrogen bonds in the human, humanized, chimeric, rabbit, chicken, or derived water around them. Examples of hydrophobic moieties from other species. include alkanes, aromatic groups, cholesterol, lipids, phos 0228. The term “antibody fragment’, as used herein, pholipids, and fatty acids. refers to a portion of an antibody, such as F(ab')2, Fab', Fab, 0233. The term “aliphatic group', as used herein, refers to Fv, slfv, diabodies, linear antibodies, fragments produced by acyclic or cyclic, non-aromatic compounds. Examples of ali a Fab expression library, anti-idiotypic (anti-Id) antibodies, phatic groups include, but are not limited to, linear or the complementarity determining region (CDR), the extracel branched alkyl chains, fatty acid chains (e.g., oleic acid), and lular domain (EDC), and epitope-binding fragments of any of long chain alkylthiols (e.g., hexanethiol). the above that immunospecifically bind to cancer cell anti 0234. The term "solid support', as used herein, refers to a gens, viral antigens or microbial antigens, single-chain anti Support that is conventionally used in organic chemistry, for body molecules, and multispecific antibodies formed from example in oligo and peptide synthesis. The term "solid Sup antibody fragments. Regardless of structure, an antibody port', as used herein, also refers to a Support that has been fragment binds with the same antigen that is recognized by used in biochemistry and biology, for example, for biopoly the full-length antibody. The term “antibody fragment also mer immobilization and purification. Examples of a solid includes isolated fragments consisting of the variable regions Support includes polystyrene Supports, polyamide Supports, of antibodies, such as the “Fv’ fragments consisting of the polyethylene glycol Supports, polyacrylic Supports, compos variable regions of the heavy and light chains and recombi ite Supports and polymers thereof. Such as polyacrylic/beta nant single chain polypeptide molecules in which light and alanine copolymer Supports, polyacrylamide/polystyrene heavy variable regions are connected by a peptide linker copolymer Supports, polyacrylamide/polyethylene glycol (scFv proteins). copolymer supports, polyethyleneglyco?polystyrene copoly 0229. The terms “nucleic acid”, “oligonucleotide', mer Supports, controlled pore glass, agarose, dextrangel, and "oligo', or “polynucleotide', as used herein, refer to a poly polysaccharide based polymers. In some cases, the term mer of nucleotides. The polymer may include, without limi “solid support' also refers to a particle that has been used in tation, natural nucleosides (i.e., adenosine, thymidine, gua biological assays, for example, or a polymeric microsphere. nosine, cytidine, uridine, deoxyadenosine, deoxythymidine, Examples of Such support include a latex microsphere, a deoxyguanosine, and deoxycytidine), nucleoside analogs polymeric particle consisting of polystyrene or copolymers of (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo styrene, poly(methyl methacrylate), polyvinyltoluene, pollu pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5-bro (2-hydroxyethyl methacrylate) and the copolymer, poly(eth mouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl ylene glycol dimethacrylate/2-hydroxyethylmetacrlate), and uridine, C5-propynyl-cytidine, C5-methylcytidine, poly(lactic-co-polycolic acid). "Solid Support' can also 7-deaZaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-OX include inorganic constructs, metals and semiconductors, oguanosine, O(6)-methylguanine, and 2-thiocytidine), Super paramagnetic composites, biodegradable constructs, chemically modified bases, biologically modified bases (e.g., and synthetic dendrimers and dendrons, such as a quantum methylated bases), intercalated bases, modified Sugars (e.g., dot, a dye-coded particle, and a magnetic-coded particle. 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hex 0235. The term “MW, as used herein refers to molecular ose), or modified phosphate groups (e.g., phosphorothioates weight. and 5'-N-phosphoramidite linkages). Nucleic acids and oli 0236. The term “small molecule', as used herein refers to gonucleotides may also include other polymers of bases hav a low molecular weight organic compound that is below 800 ing a modified backbone, such as a locked nucleic acid Da. Examples of the small molecules such as biotin, fluores (LNA), a peptide nucleic acid (PNA), or a threose nucleic acid cent labeling compound, Sugar, Sugar alcohol, metabolites, drugs, pesticides, amino acids, nucleotide, chemilluminent (TNA). compound, crosslinking reagent. 0230. The term “small interfering RNA” or “siRNA', as 0237. The term “single MW or “monodisperse', as used used herein, refers to small inhibitory double-stranded RNA herein refers to one or a collection of compounds having the molecules that induce the RNA interference (RNAi) pathway. same size and molecular weight. Natural polymers, such as siRNA generally have from about 18 to about 30 base pairs proteins, peptides, and DNA, are typically monodisperse. SA and exhibit varying degrees of complementarity to their target macromolecules are synthesized and purified from pure mRNA in the antisense strand. Some, but not all, siRNA have chiral starting materials as single MW compounds via stan unpaired overhanging bases on the 5' or 3' end of the sense dard organic synthesis techniques. SA macromolecules may strand and/or antisense strand. The term “siRNA’ includes contain mixtures of compounds that have the same MW but duplexes of two separate strands, as well as single strands that are the Stereo or regional isomers of each other. These isomers can form hairpin structures comprising a duplex region. are generated during synthesis and can be minimized by 0231. The term “polyglycerol, as used herein, refers to a choosing the right conditions or purification method. polymerized glycerol. Glycerol has the structure 0238. The term “polydisperse' or “polydispersity', as OH-CH CH(OH)—CH2—OH. A polyglycerol contains used herein refer to a collection of polymer compounds that US 2015/001 7246 A1 Jan. 15, 2015 have different sizes, shapes, or molecular weights. For peutic biopolymers. Conjugating high MW polymers to example, a polymer usually has a distribution of molecular therapeutic biopolymers may stabilize the Substances being mass over a certain range. The polydispersity index (PDI), or conjugated in circulation, reduce their immunogenicity, heterogeneity index, measures the distribution of molecular decrease antibody recognition, and increase body residence mass in a given polymer. The index is calculated based on the time. Other benefits of polymer conjugation include the pos following formula: PDI-M/M, wherein M is the weight sibility of modifying organ disposition, drug penetration by average molecular weight and M is the number average endocytosis, and new drug targeting (F. M. Veronese, M. molecular weight. Depending on the synthetic method used, Morpurgo "Bioconjugation in pharmaceutical chemistry” IL most of the synthetic polymers except peptides and oligos are Farmaco 1999, 54, 497-516). In the case of conjugating pro polydisperse. Peptides and oligos are synthesized based on teins and peptides, modification also protects the protein and standard organic synthesis techniques using pure starting peptide from proteolytic degradation in vivo. materials and are generally purified to obtain monodispersity. 0244 High MW polydisperse PEG linkers have been used to modify therapeutic proteins, antibodies, and Small toxins DETAILED DESCRIPTION OF THE INVENTION due to their hydrophilicity and biocompatibility (Veronese M. 0239 Crosslinking reagents and conjugation chemistry F. "PEGylated protein in drugs: basic science and clinical are two key parts of bioconjugation technologies. Several applications.” 2009, Birkhäuser Verlag). Examples of PEGy factors need to be considered when choosing a crosslinking lated proteins or oligonucleotides approved by the FDA are reagent for bioconjugation reactions: (i) reactive groups at the pegadamase (AdagenR), pegfilgrastim (NeulastaR), pegas termini of the linker; (ii) the length of the spacer; and (iii) the pargase (Oncaspar R), and PEG-EO (Mirera.R.). physical properties of the spacer, Such as whether it is hydro 0245 However, PEG-based linkers have several draw phobic or hydrophilic, and cleavable or biodegradable after backs. (i) The polydispersity of high MW (M) PEG mol conjugation. ecules due to the nature of polymerization chemistry. The 0240 Typical reactive groups at the termini of the linkers M/M, (number-average) value is approximately 1.01 for are pre-activated carboxylate, such as N-hydroxySuccinimide polymers with M. ranging from 2 to 10 KDa, but reaches (NHS) ester; thiol-reacting groups, such as maleimide/alkyl values up to 1.2 for higher MW polymers. The use of poly halide; and ketone or aldehyde-reacting groups, such as disperse PEG molecules to label biomolecules generates hydrazine/semicarbazide?aminooxy. Other termini groups polydisperse conjugates with Subtle differences in biological areaZide/alkyne and diene/dienophile. Biomolecules, such as properties. This result also complicates analysis of the con antibodies and proteins, usually have several same or similar jugates. A monodisperse, high M molecule is desirable, but functional groups that can interact with the terminal group of current technologies for synthesizing and purifying PEG are the crosslinking reagents. The position and degree of biomol too difficult and expensive for a commercial monodisperse ecule labeling usually varies after conjugation. Heterogeneity PEG product. Efforts have been made by scientists from of the conjugates is a major concern in drug R&D. Using a Quanta Biodesign Limited Company to develop a better pro single and pure heterobifunctional crosslinking reagent and cess for making single pure PEG (US 2010/0009902 A1, site-specific labeling can decrease the complexity. For 2010). Thus far, the highest single MW commercially avail example, Sulfhydryl is a popular functional group due to its able PEG is in the range of 1600-1700 Da (36 ethylene glycol reaction specificity and easy introduction through in vitro monomer). This process is not likely to result in a single pure cysteine mutagenesis. Sulfhydryl also plays special roles in MW PEG larger than 2000 Da due to the small MW incre specific antibody modification and conjugation. Other func ments of the monomer (one repeat unit: 44 Da). (ii) PEG is not tional groups that can be targeted include post-introduced biodegradable. The use of high M. PEG and chronic admin aldehyde, hydrazine, and azide functional groups. istration of any M, PEG may have long-term effects on 0241 Previous technologies for conjugating biomol human beings with questionable safety. (iii) Limited func ecules rely on classical spacers, such as hydrophobic alkyl tional groups are available for conjugating Small MW drugs, chains, peptides, and ethylene glycol. Many important con resulting in low drug loading. Only one or two OH groups are jugation reactions involve linking a very hydrophobic com available for drugattachment per linear PEG. Thus, noPEGy pound. Such as a hydrophobic drug, toxin, biotin, or fluores lated small MW drugs are commercially available at this cent compound, to a water soluble biomolecule, such as an point. Branched PEG and a dendrimeric structure bearing a antibody, protein, or enzyme. In most cases the reaction is few more OH groups have been introduced. (iv) Despite the performed in an aqueous buffer. The presence of a hydro hydrophilicity of the PEG linker, certain conjugates through philic linker favors the conjugation reaction and preserves the the PEG linker still lack sufficient solubility for biological stability of the final products. applications. Better hydrophilic linkers need to be developed. 0242 Hydrophilic linkers have been adapted by biochem 0246 Sugar molecules are probably the most hydrophilic ists to modulate the properties of various molecules. Such natural compounds to date due to their hydrogen bonding linkers, which are available in different motifs, have been capability (donor and acceptor). Polysaccharides have been used as agents of drug delivery, aqueous solubility enhancers linked to proteins of therapeutic interest, leading to an for hydrophobic molecules, tethers or spacers in conjugation, increased retention time in the blood, decreased immunoge encapsulating molecules in nanotechnology, and in cosmetic nicity, and the desired minimal loss of biological activity formulations. Polyethylene glycol (PEG) is one of the most (Imperiali, B, O'Connor SE, Curr. Opin. Chem. Biol. 1999,3, commonly used hydrophilic linkers and commercially avail 643-649; Sinclair A M, Elliott S. J. Pharm. Sci. 2005, 94, able. Lower MW heterobifunctional PEG compounds are the 1626-1635: Fernandes A I, Gregoriadis G. Int. J. Pharm. most frequently used compounds for linking hydrophobic 2001, 217, 215-224). Polysaccharide derivatives, such as compounds. hydroxyethyl starch (M. Orlando, Justus-Liebig Universitat 0243 High MW polymers have been used to conjugate Giesssen, 2003, p 191) and poly(sialic acid), have been used proteins, peptides, oligonucleotides, siRNA, and other thera for protein conjugation (Constantinou, A. et al. Bioconjugate US 2015/001 7246 A1 Jan. 15, 2015 15

Chem. 2008, 19,643-650; Gregoriadis G. Int. J. Pharm. 2005, purification handle (item 20, hydrazide). In a more common 300, 125-130). Due to the complexity and difficulties of sugar case, a functionalized isotopic coded mannitol (item 14) is chemistry, the polysaccharides used in Such studies tend to be used for protein footprinting. In another case, a D-mannitol is polydisperse, as in the case of PEG, and variable instructure. In addition, polysaccharides with rigid structures may result used as a spacer to link 2-aminopyridyl to a Sugar molecule in a great propensity for immunogenicity. (Galb1-4Fuc) for fluorescent assay purposes (Nishiyama K. 0247 Sugar alcohol, also known as polyol, is a hydroge et al. Chem. Pharm. Bull. 2010, 58,495-500). To the best of nated form of carbohydrate in which the carbonyl group our knowledge, none of these Small molecule functionalized (aldehyde or ketone) has been reduced to a primary or sec SA compounds have been used as a spacer for linking two or ondary OH group. Sugar alcohols occur naturally in foods more therapeutic drugs together, such as a toxin, protein, and come from plant products, such as fruits and berries. antibody, siRNA, etc. The reported SA compounds have lim Sugar alcohols are often used as ingredients in Sweeteners, for ited utility due to the fact that all of these compounds are very making polymers such as hydrogel. simple and not very efficient; in particular, no efficient het 0248. Some structures of simple functionalized low MW erobifunctional crosslinkers have been reported in the litera monomeric sugar alcohols have been reported. Table 1 lists ture. TABLE 1. Published functionalized monomeric Sugar alcohol compounds Chemical formula: X-CH)-(CHOH), CH-Y). Item if n X Y CAS if Reference 1 2 - OH —N 87691-84-7 Gen. Offen. 1983, DE 3150917 A1 19830630. 2 2 - OH —C=C-Et 29549-24-2 Tetrahedron Letters, 1990, 31(12), 1783-4. 3 2 - OH —C=CH 460348-22-5 Compt. rend. 1961,252, 751-3. 4 2 - SH —S—S-pyridyl O6OOS-81-6 Eur: Pat. Appl., 1986, EP 184361 A2 19860611. 5 2 - SH —ONH2 O3528-14-9 Bioorganicheskaya Khimiya, 1986, 12(6), 845-7. 221568-11-1 J. Am. Chem. Soc., 2010, V132(31), P10642-10644 O3722-73-2 Tetrahedron, 1985, V41 (17), P3497-509 28227–44-3 Bulletin de la Societe Chimique de France 1963, 10, 2105-13 9 2 - CONHNH – CONHNH 4461-85-2 Canadian Journal of Chemistry1956, 34, 1440-3. 10 3 - OH —N 78757-82-9 Tetrahedron Letters, 1996, 37(21), 3683–3686. 11 3 - N. —N 78757-79-4 Journal of Carbohydrate Chemistry, 2004, V23(2 & 3), P95-110 12 3 - OH —C=CH 524O3S-70-9 Tetrahedron 2003, 59(2), 155-164. 13 3 - Br —CO. NH-NH2 951O3-66-5 Acta Chemica Scandinavica, Series B: Organic Chemistry and Biochemistry 1984, B38 (7), 555-61. 14 3 - OH —CO. NH-NH2 951O3-68-7 Acta Chemica Scandinavica, Series B: Organic Chemistry and Biochemistry 1984, B38 (7), 555-61. 15 4 - OH —NH-NH2 151069-61-3 Eur: Pat. Appl., 1993, EP 545.195 A1 19930609. 16 4 - OH —N 138245-74-6 Tetrahedron: Asymmetry, 2006, V17(9), P1349-1354 17 4 - OH —NH-C(O)—CH2Br 1313868-84-6 J. Mol. Bio 2011, 4.09(4), 483-495. 18 4 - OH —NH-C(O)—NH2 885O24-84-0 Fr. Demande, 2006, FR 2877221A1 20060505. 19 4 - N. —N 52868-75-4 Journal of Carbohydrate Chemistry, 2004, V23(2 & 3), P95-110 2O S OH - C(O)NHNH2, 130538-63-5 J. Am. Chem. Soc. 1946, V68, P1509-10 most of the functionalized monomeric Sugar alcohols that 0249 Synthetic high MW SA molecules are rare in the may be used for the conjugation published in the literature (if literature. A mannitol dimer has been synthesized for use as the compound has more CAS numbers or references, only one liquid crystal (Akiyama, H. et al. Journals of Materials is listed here). The data were obtained by extensively search Chemistry, 2009, 19, 5956-5963). New derivatives of disor ing for the formula X CH (CHOR), CH Y, where bity1-amine have been reported (Pol. 2010, PL 206420 B1 n is 2 to 12, R can be H, Me, or any other protecting group, and 20100831). However, none of these possess any useful X and Y can be anything. Compounds containing only NH crosslinker groups. On the other hand, nature produces some COOH, SH, OH, Br, or NO, were excluded from the table of the SA polymers. For example, the bacterial cell wall because these compounds require further activation before associated teichoic acids predominantly contain D-ribitol they can be used for conjugation. A total of approximately 60 residues interconnected by phosphodiester linkages. Because compounds containing any kind of functional group were of their location, Fekete, Aniko et al. Synthesized octa- and found for monomeric Sugar alcohol. The purpose of synthe dodecamers of D-ribit 1-1-phosphate for the purpose of devel sizing Such compounds falls mainly into the following cat oping a new vaccine (Carbohydrate Research, 2006, V341 egories: 1) as an initiator or starting material for polymeriza (12), P2037-2048). The polymer is coupled to a vaccine car tion, Such as making polyhydroxylated nylon 6 or other rier, bovine serum albumin, through a keto handle and will be polymers containing hydroxyl groups; 2) as starting material used as a single-component experimental vaccine. Nothing in for a small molecule drug, in most cases becoming a part of the literature has suggested that Such oligomers can be used to the drug with little resemblance to the sugar alcohol; or 3) as modulate the pharmacokinetic and pharmacodynamic prop pure, synthetic method development for functional group erties of other therapeutic drugs or as carriers for drug deliv conversion. In rare cases the functional group is used as a ery in vivo. US 2015/001 7246 A1 Jan. 15, 2015

0250 Other less relevant sugar alcohol structures, most of which are polymers, can also be found in WO2010014678, PL206420, WO2010/134476, WO2010134476, JP200924.9500, FR2906245, WO2009053596, FR2906245, WO2004025297, WO2001005224, FR2701949, WO9421595, EP536939, U.S. Pat. No. 4,172,094, EP675101A1, polyglycerol U.S. Pat. No. 2,520,670, polyhy droxy compound U.S. Pat. No. 2,520,671, and polyhydric alcohol U.S. Pat. No. 2,532,036. 0251 Thus, a critical need exists for hydrophilic crosslinking reagents, single high MW compounds, and high loading carriers in the biotechnology and pharmaceutical fields. The idea of using a hydrophilic Sugar alcohol as a backbone to build up crosslinking reagents for linking differ ent class of drugs, a single MW SA macromolecule to site specifically label therapeutic proteins, and enable such prac tice can revolutionize the whole pharmaceutical industry. 0252. The present invention provides for a conjugate hav ing a structural formula selected from the group consisting of 0260 each G' is independently selected from the group M-(L-B). Formula (I), and consisting of NR, O, and S; B-(L-M), Formula (II) 0261) each G’ is independently selected from the group consisting of O and S. 0253 wherein, 0262 each G is independently selected from the group 0254 each M is independently selected from the group consisting of S, O, NR, and SO; consisting of a protein, an enzyme, an antibody, an anti 0263 each G' is independently selected from the group body fragment, a polypeptide, an oligonucleotide, an consisting of O and NR; oligonucleotide analog, and a polysaccharide; 0264 each R is independently selected from the group consisting of a bond, C-C alkyl, -(CH2CH2O). 0255 each B is a single MW modified sugar alcohol 10-, -(CH2CH2O). CH , optionally substi polymer, comprising from 2 to about 2000 Sugar alcohol tuted alicyclyl, heteroalicyclyl, aryl, a peptide, and a monomer(s): peptidomimetic oligomer, 0265 each R is independently selected from the group 0256 each monomer has from 3 to about 14 —OR' consisting of hydrogen, C-C alkyl, —(OCH2CH2). groups: alicyclyl, and heteroalicyclyl, wherein any ring in R is 0257 each R" is independently selected from the group optionally substituted. consisting of hydrogen, C-Cs alkyl, benzoyl, acetyl, 0266) each R is independently C-Cs alkyl; and, benzyl, C-C alkyl silyl cyclic ortho ester, actinide, 0267 u is an integer from 1 to about 20. acetate, tetrahydropyranyl, tetrahydrofuranyl, and tet 0268. The present invention also provides for a conjugate rahydrothiofurany; having a structural formula: 0258 each L is independently selected from the group (M2-L)-B Formula (III) consisting of a Rand, a structure of V. R. V. , 0269 wherein, 0270 each M is independently selected from the group 0259 each V and V are independently selected from consisting of a metabolite, a fluorescent compound, a the group consisting of a Diels-Alder adduct, a 13 chemiluminescent compound, a mass tag, a chro dipolar adduct, —C(=G)-G'-, -G-C(=G)-, -G -, -G'- mophore, biotin, a toxin, a drug, a chemotherapeutic C(=G)-G-, - S S , —S (CH), S(=O) , agent, a cytotoxic agent, an immunosuppressive agent, a S(=O) (CH2) S , S(=O). N(R) , diagnostic agent, a radioligand, and a small molecule, N(R) S(=O). , C(=O) NH NH 0271 each B is a single MW modified sugar alcohol polymer, comprising from 2 to about 2000 Sugar alcohol monomer(s): (0272 each monomer has from 3 to about 14 —OR' groups: 0273 each R" is independently selected from the group consisting of hydrogen, C-C alkyl, benzoyl, acetyl, benzyl, C-C alkyl silyl cyclic ortho ester, actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl, and tet rahydrothiofurany; 0274 each L is independently selected from the group consisting of a Rand, a structure of V. R. V. , US 2015/001 7246 A1 Jan. 15, 2015 17

0275 each V and V are independently selected from 0284. The present invention also provides for a conjugate the group consisting of a Diels-Alder adduct, a 13- having a structural formula selected from the group consist dipolar adduct, —C(=G)-G'-, -G-C(=G)-, -G -, -G'- ing of (M1)-L-(B-(L-M2)), Formula (IV) (M-L)-(B-(L-M2)), Formula (V) and M1-(L-B-(L-M-2)), Formula (VI) 0285 wherein, 0286 each M is independently selected from the group consisting of a protein, an enzyme, an antibody, an anti body fragment, a polypeptide, avidin, Streptavidin, an oligonucleotide, an oligonucleotide analog, and a polysaccharide; 0287 each M is independently selected from the group consisting of a metabolite, a fluorescent compound, a chemiluminescent compound, a mass tag, a chro mophore, biotin, a toxin, a drug, a chemotherapeutic agent, a cytotoxic agent, an immunosuppressive agent, a diagnostic agent, a radioligand, and a small molecule: 0288 each B is a single MW modified sugar alcohol polymer, comprising from 2 to about 2000 Sugar alcohol monomer(s): (0289 each monomer has from 3 to about 14 —OR' groups: 0290 each R" is independently selected from the group consisting of hydrogen, C-C alkyl, benzoyl, acetyl, benzyl, C-C alkyl silyl cyclic ortho ester, actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl, and tet rahydrothiofurany; 0291 each L is independently selected from the group consisting of a Rand, a structure of V. R. V. , 0292 each V and V are independently selected from the group consisting of Diels-Alder adduct, a 1,3-dipolar adduct, —C(=G')-G-, -G-C(=G')-, -G-, -G-C (0276 each G' is independently selected from the group (G)-G-, - S S , S (CH), S(=O) , consisting of NR, O, and S; S(=O)—(CH2) S , S(=O) N(R) s N(R) S(=O). , C(=O) NH NH (0277 each G’ is independently selected from the group CH , C(=O) NH N=CH- CH N. consisting of O or S; NH C(=O) , CH-NH NH C(=O) , (0278 each G is independently selected from the group N(R) S(=O), N(R) C(=O) NH CH consisting of S, O, NR, and SO2: (CHSH) , -N=CH-, -NH-CH , -NH C (=O)-CH C(=O) NH-, CH-N-G-, (0279 each G' is independently selected from the group CH, NH-G-, -G -NH CH, , -G-N=CH-, consisting of O or NR; —C(=NH) NH , —NH CO NH)—, 0280 each R is independently selected from the group - O P(=O)(O) NH NH P(=O)(O) consisting of a bond, C-C alkyl, -(CH2CH2O). O , —CH-CH(NH)—CH, S , —S CH 10—, —CH2CH2O). CH , optionally Substituted CH(NH)-CH - O P(=O)(O)- O - O P alicyclyl, heteroalicyclyl, aryl, a peptide, and a peptido (=O)(S)-O-, - O P(—S)(S)-O , mimetic oligomer, (0281 each R is independently selected from the group consisting of hydrogen, C-C alkyl, —(OCH2CH2). alicyclyl, and heteroalicyclyl, wherein any ring in R is optionally substituted. 0282 each R is independently C-C alkyl: H. :-- 0283 q is an integer from 1 to about 100. US 2015/001 7246 A1 Jan. 15, 2015 18

-continued apoptotic agents, Vinca alkaloids, paclitaxel, maytansine, calicheamicin, and dolastatins. 0308 M may be an antibody. More preferably, M is a fully human antibody. 0309 Optionally, M may be an antibody specific for an antigen selected from the group consisting of tumor-associ ated antigens, antigens associated with pancreatic cancer, antigens associated with malignant disease, antigens associ ated with autoimmune disease, antigens associated with immune dysfunction disease, antigens associated with leuke mia, antigens associated with neurological disease, antigens against transmembrane activator, and an antigen against CAML-interactor. 0310 M may also be an antibody specific for an antigen , - he selected from the group consisting of CA125, CA 15-3, CA19-9., L6, Lewis Y. Lewis X, alpha fetoprotein, CA 242, 0293 each G' is independently selected from the group placental alkaline phosphatase, prostate specific antigen, pro consisting of NR, O, and S; static acid phosphatase, epidermal growth factor, MAGE-1, (0294 each G’ is independently selected from the group MAGE-2, MAGE-3, MAGE-4, anti-transferrin receptor, p97, consisting of O and S. MUC1-KLH, CEA, gp100, MART1, IL-2 receptor, CD4, (0295) each G is independently selected from the group CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, consisting of S, O, NR, and SO2: CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, 0296 each G' is independently selected from the group CD54, CD66a-d, CD67, CD74, CD79a, CD80, CD126, consisting of O and NR; CD138, CD154, B7, MUC1, LALI, HM1.24, HLA-DR, tena 0297 each R is independently selected from the group scin, VEGF, P1GF, ED-B fibronectin, oncogenes, oncogene consisting of a bond, C-C alkyl, -(CH2CH2O). products, necrosis antigens, T101, TAG, IL-6, MIF, TRAIL 10—, —(CH2CH2O)o CH2—, optionally Substi R1 (DR4), TRAIL-R2 (DR5), human chorionic gonadotro tuted alicyclyl, heteroalicyclyl, aryl, a peptide, and a pin, mucin, P21, MPG, and Neu oncogene product. peptidomimetic oligomer, 0311 At least one M may comprise a radioactive isotope. 0298 each R is independently selected from the group 0312. At least one M may be a therapeutic protein or consisting of hydrogen, C-C alkyl, —(OCH2CH2). polypeptide. Preferably, the amino acid sequence of the thera alicyclyl, and heteroalicyclyl, wherein any ring in R is peutic protein contains at least 80% sequence homology to optionally substituted. the wild-type therapeutic proteins selected from the group (0299 each R" is independently a C-Cs alkyl; consisting of granulocyte macrophage colony stimulating (0300 u is an integer from 1 to about 100 factor, interferon, interferon alpha-2a, interferon alpha-2b. 0301 q is an integer from 1 to about 100 and interleukin, interleukin-2, erythropoietin, growth hormone, 0302 k is 0 or an integer from 1 to about 20. human growth hormone, apomyoglobin, asparaginase, leptin, 0303 Preferably, B comprises from 4 to about 2000 sugar serum proteins, human chorionic gonadotropin, insulin, fol alcohol monomers. licle stimulating hormone, luteinizing hormone, urate oxi 0304 Preferably, u is an integer from 1 to about 10. dase, adenosine deaminase, antibody fusion proteins, and 0305 Optionally, M is selected from the group consisting factor VII. More preferably, the amino acid sequence of the of an antibody and an antibody fragment, and M is a chemo therapeutic protein contains at least 90% sequence homology therapeutic drug, q is 1., u is an integer from 1 to about 20, and, to the wild-type therapeutic proteins selected from the group k is an integer from 1 to about 10. More preferably, in this consisting of granulocyte macrophage colony stimulating embodiment k is 1, and u is an integer selected from 1 to about factor, interferon, interferon alpha-2a, interferon alpha-2b. 8 interleukin, interleukin-2, erythropoietin, growth hormone, 0306 The sugar alcohol monomers may be linked human growth hormone, apomyoglobin, asparaginase, leptin, together by an ether linkage. serum proteins, human chorionic gonadotropin, insulin, fol 0307. In one aspect, M may a chemotherapeutic agent. In licle stimulating hormone, luteinizing hormone, urate oxi this aspect, M is a chemotherapeutic agent used in anticancer dase, adenosine deaminase, antibody fusion proteins, and therapy. Most preferably, M. maybe selected from the group factor VII. consisting of adrenocortical Suppressants, antimetabolites, 0313 At least one M may be selected from the group alkylating agents, alkyl Sulfonates, antibiotics, antimitotics, consisting of an oligonucleotide and siRNA. anthracyclines, anti-angiogenic agents, camptothecins, 0314. The present invention also provides for a conjugate COX-2 inhibitors, CPT-11, doxorubicin, doxorubicin ana having a structural formula selected from the group consist logs, enzyme inhibitors, endostatin, epipodophyllotoxins, ing of ethylenimine derivatives, folic acid analogs, gemcitabine, HDAC inhibitors, heat shock protein (HSP)90 inhibitors, hor S-(L-B-(L-M1)), Formula (VII), mone antagonists, methotrexate, methyl hydrazine deriva tives, mTOR inhibitors, nitrosoureas, nitrogen mustards, S-(L-B-L-(M)), Formula (VIII), pyrimidine analogs, purine analogs, platinum coordination S-(L-B-(L-M-2)), Formula (IX), and complexes, Substituted ureas, SN-38, taxols, triaZenes, tax anes, tyrosine kinase inhibitors, proteosome inhibitors, pro S-(L-B-L-(M2)), Formula (X) US 2015/001 7246 A1 Jan. 15, 2015 19

0315 wherein, 0324) each G' is independently selected from the group 0316 S comprises a solid support; consisting of NR. O. and S; 0317 each M is independently selected from the group 0325 each G’ is independently selected from the group consisting of a protein, an enzyme, an antibody, an anti consisting of O and S. body fragment, a polypeptide, avidin, Streptavidin, an 0326) each G is independently selected from the group oligonucleotide, an oligonucleotide analog, a polysac consisting of S, O, NR, and SO2: charide; 0318 each M is independently selected from the group 0327 each G' is independently selected from the group consisting of a metabolite, a fluorescent compound, a consisting of O and NR; chemiluminescent compound, a mass tag, a chro 0328 each R is independently selected from the group mophore, biotin, a toxin, a drug, a chemotherapeutic consisting of a bond, C-C alkyl, -(CH2CH2O). agent, a cytotoxic agent, an immunosuppressive agent, a 10-, -(CH2CH2O). CH , optionally substi diagnostic agent, a radioligand, a small molecule; tuted alicyclyl, heteroalicyclyl, aryl, a peptide, and a 0319 each B is a single MW modified sugar alcohol peptidomimetic oligomer, polymer, comprising from 2 to about 2000 Sugar alcohol 0329 each R is independently selected from the group monomer(s): consisting of hydrogen, C-C alkyl, —(OCH2CH2). 0320 each monomer has from 3 to about 14 —OR' alicyclyl, and heteroalicyclyl, wherein any ring in R is groups: optionally substituted. 0321) each R" is independently selected from the group 0330 each R is independently a C-Cs alkyl: consisting of hydrogen, C-Cs alkyl, benzoyl, acetyl, 0331 u is an integer from 1 to about 500; and, benzyl, C-C alkyl silyl cyclic ortho ester, actinide, 0332 k is 0 or an integer from 1 to about 20. acetate, tetrahydropyranyl, tetrahydrofuranyl, and tet 0333. In this embodiment, B may be from 3 to about 1000 rahydrothiofurany; Sugar alcohol units. 0322 each L is independently selected from the group 0334. In this embodiment, S may be selected from the consisting of a R and, a structure of V. R. V. , group consisting of polystyrene Supports, polyamide Sup 0323 each V and V are independently selected from ports, polyethylene glycol Supports, polyacrylic Supports, the group consisting of a Diels-Alder adduct, a 13 polyacrylic/beta-alanine copolymer Supports, polyacryla dipolar adduct, —C(=G)-G-, -G-C(=G)-, -G-, -G - mide/polystyrene copolymer Supports, polyacrylamide/poly ethylene glycol copolymer supports, polyethyleneglycof polystyrene copolymer Supports, controlled pore glass, agarose, dextrangel, polysaccharide based polymer, a poly meric microsphere, a latex microsphere, a polymeric particle consist of polystyrene, a polymeric particle consist of copoly mers of Styrene, poly(methyl methacrylate), polyvinyltolu ene, poly(2-hydroxyethyl methacrylate), the copolymer of poly(2-hydroxyethyl methacrylate), poly(ethylene glycol dimethacrylate/2-hydroxyethylmetacrlate), poly(lactic-co polycolic acid), inorganic constructs, metals, semiconduc tors, Super paramagnetic composites, biodegradable con structs, synthetic dendrimers, dendrons, a quantum dot, a dye coated particle, and a magnetic coated particle. Optionally, S is an agarose bead. Alternatively, S may be a magnetic coated particle. 0335. In all of the above mentioned embodiments, B may be a modified Sugar alcohol polymer, comprising from 2 to about 2000 Sugar alcohol monomer(s) B", and each sugar alcohol monomer being bound to one or more Sugar alcohol monomers through a linking group W formed by a reaction between the X,Y or Z portion of one monomeric unit with the X, Y or Z of another monomeric unit; 0336 wherein, for each B", independently, has the chemi

cal structural of Formula XI

(XI) US 2015/001 7246 A1 Jan. 15, 2015 20

0337 each of n and p is independently selected from 0 and an integer selected from 1 to about 12, and n+p is between 3 and 12; 0338 r is 0 or 1: 0339 each bond represented by . is a single or a double bond; 0340 Q is selected from the group consisting of —O, —N-O-L-M, —N-O-L-M =N-O-L-S, NH O-L-S, NH-O-L-M, and NH-O-L-M, 0341 each of X, Y and Z., when bound to M. M. or S, is a linker V. (0342 each of X, Y and Z., when not bound to S or M, M, is a functional group independently selected from the group consisting of -OH, -J.-RJ, —C(=O)-J, C(=O)-CH-J, -NH C(=O) CH-J, OR, —OR, OR. -O-Mesyl, -O-Tosyl, -NH C (=O)—CH2—O-Mesyl, - NH CO O)—CH2—O- Tosyl, -SH, -S-S-tButyl, - SR", -SR,-S-S- R. S(=O).-J., NH, -NHR, N(R)R,

NH, -NH C(=S) NH NH, -toluenesulfonyl hydrazide, -R NH-C(=NH) NH, a benzophenone, an aryl diazonium, a diazoalkane, a dia Zoacetyl, an anthraquinone, a diazirine, an optionally substituted trifluoromethylphenyldiazirine, a diene, a dienophil, a 1,3-dipole, a dipolarophile, an alkene, a ketene, an olefin, an alkene with allylic hydrogen, a dicarbonyl group, an epoxide, an oxirane, an organosi lane, a phosphonium group, an ester, an anhydride, a carbonate group, a glyoxal, —C(=NH)-O-R, a hydroxymethyl phosphine derivative, an ethyl vinyl, a 0351 wherein: maleimide, a vinylsulfone, anallyl Sulfone, a thioester, a cisplatin derivative, an aziridine, an acryloyl group; O352 each G' is independently selected from NR. O. (0343 each R is independently selected from the group and S; consisting of hydrogen, C-C alkyl, alicyclyl, heteroali cyclyl benzyl or aryl, wherein any ring in R is option 0353 each G’ is independently O or S; ally substituted; 0354 each G is independently selected from S. O. (0344 each R is independently selected benzoyl, NR, and SO; acetyl, benzyl, C-C alkyl silyl cyclic ortho ester, actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl, 0355 each G' is independently O or NR; and tetrahydrothiofuranyl: 0356) each R is independently selected from a bond, (0345 each R" is independently selected from the group C-C alkyl, -(CH2CH2O)o , —(CH2CH2O). consisting of trity1, MMT, and DMT: lo—CH2—, optionally substituted alicyclyl, heteroali (0346) each R is independently selected from the group cyclyl, aryl, a peptide, and a peptidomimetic oligomer; consisting of 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophenyl, 4-nitrophenyl, 3-car 0357 each R is independently selected from hydrogen, boxy-4-nitrophenyl, and 2,4-dinitrophenyl: C-Cs alkyl, —(OCH2CH2), optionally Substituted 0347 each J is independently selected from the group alicyclyl, and optionally substituted heteroalicyclyl; consisting of Cl, Brand I 0358 each R is independently C-Cs alkyl: (0348 each of L', L, and L is independently a R or -R V R *, wherein: 0359 each R is a bond or -CH : 0349 66: represents a portion of L', L, and Lbound to 0360 and X, Y. S. Mor M, or a Z, respectively; 0350 each W and Vareindependently selected from the 0361 at least in one of the B' unit each -L-Z portion is group consisting of a Diels-Alder adduct, a 1,3-dipolar —OR'; wherein each R" is independently selected from adduct, —C(=G')-G-, -G-C(=G')-, -G-, -G-C the group consisting of hydrogen, C-C alkyl, benzoyl, (=G)-G'-, - S S , —S (CH), S(=O) , acetyl, benzyl, C-C alkyl silyl cyclic ortho ester, S(=O) (CH2) S , S(=O) N(R) s actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl, N(R) S(=O). , C(=O) NH NH and tetrahydrothiofurany. US 2015/001 7246 A1 Jan. 15, 2015

0362 Optionally, B has the chemical structural of Formula 0373 each B1, independently, has the chemical struc XII: tural Formula XIV:

(XII)

0363 wherein, represents a bond to L'; and

represents a bond to L. represents a bond to M, M, or S; 0374. In another aspect, present invention also provides 0364 each of L', when bound to M, M, or S, is a linker for a single MW compound having a linear, branched or V macrocyclic multimer Sugar alcohol comprising three or more monomeric Sugar alcohol units B'; and 0365 when k is 0, 0375 each monomeric sugar alcohol unit is bound to one or more other monomeric units through a linking group W formed by a reaction between the X, Y or Z. 2 portion of one monomeric unit with the X, Y or Z of another monomeric unit; 0376 wherein C, 0377 each B" has the chemical structural Formula XV:

represents Y, when k is an integer selected from 1 to about 20, (XV) ck represents a bond to M. M. or S. 0366 Alternatively, B may have the chemical structural 0378 wherein for each B', independently: Formula XIII: 0379 each of n and p is independently selected from 0 * I-L4-(B' W), B-Li-2 (XIII) and an integer selected from 1 to about 12; and 0380 n+p is between 1 and 12: 0367 wherein: 0381 r is 0 or 1: 0368 “1” represents a portion of the L'bound to S. M1 0382 each of X, Y and Z is independently selected from or M2: 0369 each of L', when bound to M1, M2 or S, is a linker V 0370 s is 0 or an integer independently selected from 1 to about 500; 0371 when k is 0, L4-2 represents Y; and 0372 when k is an integer selected from 1 to about 20, “*2” represents a portion of the L' bound to M1, M2, or S and US 2015/001 7246 A1 Jan. 15, 2015 22

(-NH2) NH, a benzophenone, an aryl diazonium, a dia -continued Zoalkane, a diazoacetyl, an anthraquinone, a diazirine, an optionally substituted trifluoromethylphenyldiazirine, a diene, a dienophil, a 1,3-dipole, a dipolarophile, an alkene, a ketene, an olefin, an alkene with allylic hydrogen, a dicarbo nyl group, an epoxide, an oxirane, an organosilane, a phos phonium group, an ester, an anhydride, a carbonate group, a glyoxal, —C(=NH)—O R, a hydroxymethylphosphine ,--- derivative, an ethyl vinyl, a maleimide, a vinylsulfone, an allyl Sulfone, a thioester, a cisplatin derivative, an aziridine, 0391 wherein: an acryloyl group; 0392 each G' is independently selected from NH, O, 0383 each Risindependently selected from hydrogen, and S; C-Cs alkyl, alicyclyl, heteroalicyclyl, benzyl or aryl, 0393 each G’ is independently O or S; wherein any ring in R is optionally substituted; 0394 each G is independently selected from S. O. (0384 each R is independently selected from benzoyl, NR, and SO: acetyl, benzyl, C-C alkyl silyl cyclic ortho ester, 0395 each G' is independently O or NR; actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl, 0396 each R is independently selected from a bond, and tetrahydrothiofuranyl: C-C alkyl, -(CH2CH2O)o , —(CH2CH2O). lo—CH2—, optionally substituted alicyclyl, heteroali (0385) each R" is independently selected from trityl, cyclyl, aryl, a peptide, and a peptidomimetic oligomer; MMT, and DMT: 0397) each Risindependently selected from hydrogen, 0386 each R is independently selected from 2-pyridyl, C-C alkyl, -(OCH2CH), alicyclyl, heteroalicy 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitro clyl, wherein any ring in R is optionally substituted; phenyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, and 0398 each R is independently C-Cs alkyl; 2,4-dinitrophenyl; 0399 each R is a bond or -CH ; and, 0387 each J is independently selected from Cl, Brand (0400 at least in one of the B' unit each-L-Zportion is I —OR'; wherein each R" is independently selected from the group consisting of hydrogen, C-C alkyl, benzoyl, 0388 each of L', L, and L is independently a R or acetyl, benzyl, C-Cs alkyl silyl cyclic ortho ester, -R V R *, wherein: actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl, 0389) “*” represents a portion of L', L, and Li and tetrahydrothiofurany; at least in one of the B' unit, bound to X, Y, or a Z, respectively; n+p+r is greater than 1. 0390 each V and W is independently selected from the 04.01 Preferably, the compound has a molecular weight group consisting of a Diels-Alder adduct, a 1,3-dipolar from about 5 kDa to about 500 kDa. More, preferably, the adduct, —C(=G')-G-, -G-C(=G')-, -G-, -G-C compound has a the molecular weight of the compound is (G)-G-, - S S , S (CH), S(=O) , from about 10 kDa to about 100 kDa. Most preferably, the S(=O) (CH2) S , S(=O). N(R) , compound has a molecular weight is from about 10 kDa to - N(R) S(=O) , C(=O) NH NH about 60 kDa. CH-, - C(=O) NH N=CH-, CH N. 0402 Preferably, the compound has a purity of greater NH C(=O) , CH-NH NH C(=O) , 90%. More preferably, the compound has a purity of greater N(R) S(=O), N(R) C(=O) NH CH than 95%. (CHSH) , -N=CH-, - NH-CH , -NH C 0403. Most preferably, the compound has a molecular (=O)-CH C(=O) NH-, CH-N-G-, weight from about 10 kDa to about 60 kDa and the compound CH NH-G-, -G-NH-CH , -G-N=CH-, has a purity of greater 95%. —C(=NH) NH , —NH C(=NH) , 04.04 The compound may have a W selected from the - O P(=O)(O) NH NH P(=O)(O) group consisting of —S— —O— —NH-, —C(=O) O , —CH2—CH(NH)—CH2—S —S CH NH —NHC(=O)— —S(=O)— —S(=O) , CH(NH) CH-, - O P(=O)(O)-O-, - O P —P(=S),O , and - P(=S)(=O)C -. (=O)(S)-O-, and - O P(=S)(S)-O , 04.05 Optionally the compound has a W selected from the group consisting of—S— —O— —S(=O)— —S(=O) , P(=S)O—, and - P(=S)(=O)C)—. More prefer ably, W is —O—. 0406 Preferably, at least one of X,Y, or Z of the compound may be selected from —O NH, -N=C=S, N-C-O, C=C R, N-N-N , SR, 23-7- —S S R. —C(=O)—CH-J, a diene, a dienophil, a ketone, an aldehyde, a 1,3-dipole, a dipolarophile, an alkene, a ketene, an olefin, an alkene with allylic hydrogen, trifluo romethyl phenyldiazirine, N-hydroxylsuccimidyl ester, and maleimide; (0407 each R may be independently selected from Fr. hydrogen, C-C alkyl, alicyclyl, heteroalicyclyl, benzyl and aryl, wherein any ring in R is optionally substituted; US 2015/001 7246 A1 Jan. 15, 2015

(0408) each R may be independently selected from -continued 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-py NHCI ridyl, 2-nitrophenyl, 4-nitrophenyl, 3-carboxy-4-nitro phenyl, and 2,4-dinitrophenyl; and each J is indepen o1 dently selected from Cl, Br, and I. 04.09 Optionally, for the compound, each of L', Li may be —CH2—, each Limay be a bond, and each Z may be —OR', 0416) The compound may also have each -L-Zbe –OH. wherein R' is independently selected from the group consist ing of hydrogen, C-C alkyl, benzoyl, acetyl, benzyl, C-Cs 10417. The compound may also have each L' and Libe a alkylsilyl cyclic ortho ester, actinide, acetate, tetrahydropy —CH2—. ranyl, tetrahydrofuranyl, and tetrahydrothiofurany 0418. The compound may also have each r be 0. 0410 Optionally, for the compound, each X may be 0419. The compound may have at least one X, and at least —O—(C-C alkyl); at least one Y may be —N—N'—N. one Y independently selected from —OH, - OC-C alkyl, —O NH, alkyl, - NH CO =O). CHCH-maleimide, S–S R. O. NH2, C(=O) OH, N-hydroxysuc S S R, NH-(C=O) CH Br, NH C cimide, NH C(=O). CHCH-maleimide, NH C (=O) CH-CH S S R, -SH, S C(=O) (=O)—CH2—Br. CH,

O

N , and H H N S-S N NN O O CF3: N --~~ryH -- S-S O and—S C(=O)—CH; and wherein X and Y are different. Preferably, each -L-Z is –OH. More preferably each -L-Z and may be –OH and each L', Li may be —CH2—, Most pref 0411 each R is independently selected from 2-pyridyl, erably, each-L-Zis-OH, each L', Li may be —CH2—and 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitro each r is 0. phenyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, and 0420. The compound may have at least one X,Y, or Z may 2,4-dinitrophenyl. be a vinylsulfone group or has the formula: 0412 Optionally, each -L-Z portion of the compound may be —OR' wherein R' may be independently selected from the group consisting of hydrogen, C-C alkyl, benzoyl, R10 acetyl, benzyl, C-Cs alkylsilyl cyclic ortho ester, actinide, O V acetate, tetrahydropyranyl, tetrahydrofuranyl, and tetrahy S drothiofurany. R 111Y V 0413 Optionally, for the compound, each L' and L. may \, be —CH2—. 0414 For the compound, each r may be 0. wherein R' is an electron withdrawing group; and R'' is an 0415. The compound may have at least one X and at least optionally substituted aryl. one Y that are the same and may be selected from N-hydrox 0421 Optionally, the multimer of the compound may be ySuccimidyl, —NH CO =O). CHCH-maleimide, linear and have the following structure XVI - N=N*—N, O NH, -C=C-C-C alkyl, (XVI)

US 2015/001 7246 A1 Jan. 15, 2015 24

0422 wherein V is an integer from 2 to about 2000. More preferably, each-L-Zportion of the compoundis —OR" and has the following structure XXI

(XXI)

wherein 0424 Optionally, the compound may be a multimer which R" is independently selected from the group consisting of is cyclic having the structural formula XIX hydrogen, C-Cs alkyl, benzoyl, acetyl, benzyl, C-C alkyl silyl, cyclic ortho ester, actinide, acetate, tetrahydropyranyl. tetrahydrofuranyl, and tetrahydrothiofurany. Even more pref (XIX) erably, each ris 0 and the multimer has the following structure XVII

(XVII) 1 L L2 L L2 X w1. ORI Y n-p OR n"p /, whereint is an integer from 3 to about 2000, each p may be wherein R' is independently selected from the group consist independently selected from 1 to about 12. ing of hydrogen, C-C alkyl, benzoyl, acetyl, benzyl, C-Cs 0425 Optionally, the compound may be a multimer which alkylsilyl cyclic ortho ester, actinide, acetate, tetrahydropy is a branched macro Sugar alcohol compound having the ranyl, tetrahydrofuranyl, and tetrahydrothiofurany. Even structural formula XX: more preferably, the compound has the following structure XVIII (XX) Y

OH (XVIII) pi L L2 W

ORI OH ORI pi

OH W pal W. X pi pi p Y W OH" wherein R' is independently selected from the group consist ing of hydrogen, C-C alkyl, benzoyl, acetyl, benzyl, C-Cs alkylsilyl cyclic ortho ester, actinide, acetate, tetrahydropy O ranyl, tetrahydrofuranyl, and tetrahydrothiofurany. Even more preferably, the compound wherein W is selected from W the group consisting of—S— —O— —NH , —C(=O) O 0423 Optionally W may be selected from the group con sisting of —S—, O—, S(=O) , S(=O) , Y —P(=S),O , and - P(—S)(=O)C) . Preferably, W is —O—. Optionally, X and Y is not the same. US 2015/001 7246 A1 Jan. 15, 2015 wherein each n and p is an integer independently selected 0438 Optionally, the monomeric sugar alcohol may have from 0 to 12, and n+p is between 2 and 12 0439 X be C(=O) OH: ris 0, and Y be indepen dently selected from NH-R', -NH-Fmoc, NH each n is an integer independently selected from 1 to 12; Boc, S. S. R. S. S. R. S S-tButyl, each V is an integer independently selected from 0 to 2000. —O NH-Fmoc, —O N-(Boc), and —O N(-ph 0426. The present invention also provides, a monomeric thalimidyl); wherein; sugar alcohol having the chemical structural Formula XXVI: 0440 each R is independently selected from 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitro phenyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, and

(XXVI) 2,4-dinitrophenyl; 0441 each R" is independently selected from trityl, MMT, and DMT; and when r is 1, then Y is OR' wherein; 0442 each R" is independently selected from the group consisting of hydrogen, C-C alkyl, benzoyl, acetyl, benzyl, C-C alkyl silyl cyclic ortho ester, actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl, and tet 0427 each of n and p may be independently selected rahydrothiofurany. from 0 and an integer selected from 1 to about 12; and 0443) Optionally, the monomeric sugar alcohol may have n+p is between 2 and 12: X' be a phosphoramidite; 0428 r is 0 or 1: 0444 when r is 0, Y be independently selected from 0429 each of X may be independently selected from -S-S-(C-C alkyl)-OR7, S-S-R, NH-TFA, OH, -J, —C(=O)-CH-J, OR, -S-S-R, - NH R", wherein NH-C(=O). CHCH S S R, —S—C 0445 each R" is independently selected from trityl, (=O)-CH, C(=O)H, -C(=O) R, C(=O) MMT, and DMT OH, -C=C R, N=N*—N, O NH, 0446 each R is independently selected from 2-py —O NH-Fmoc, —O NH-Boc, —O N-(Boc), ridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, —O N(-phthalimidyl), —C(=O)—NH NH, a 2-nitrophenyl, 4-nitrophenyl, 3-carboxy-4-nitrophe phenol group, an optionally Substituted trifluorometh nyl, and 2,4-dinitrophenyl. ylphenyldiazirine, a diene, a dienophil, a 1,3-dipole, a 0447. The monomeric sugar alcohol may have X and Y' dipolarophile, an alkene, a ketene, an olefin, an alkene not be the same. More preferably, at least one of X" and Y is with allylic hydrogen, an optionally substituted N-hy selected from the group consisting of —O—NH2, —S-S- droxysuccinimide ester, a imidoester, a maleimide; R. NH C(=O). CHCH S S R, an optionally Substituted N-hydroxysuccimide ester group, a optionally 0430 each of Y may be independently selected from substituted trifluoromethylphenyldiazirine, a maleimide —S—S-tButyl, SR7, S S R, NH C group; wherein each R is independently selected from 2-py (=O). CHCH S SR, NHR'. - NH-Fmoc, ridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitro NH-Boc, —O NH, O. NH-Fmoc, O. NH Boc, —O N-(Boc) —O N(-phthalimidyl), an phenyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, and 2,4- optionally substituted trifluoromethylphenyldiazirine, dinitrophenyl an optionally Substituted N-hydroxySuccinimide ester, a 0448. The monomeric sugar alcohol may have a chemical imidoester, a maleimide; formula of 0431 each R' may be independently selected from the group consisting of hydrogen, C-C alkyl, benzoyl, acetyl, benzyl, C-C alkyl silyl cyclic ortho ester, actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl, and tetrahydrothiofurany; Nullpi 0432 each R may be independently selected from hydrogen, C-C alkyl, alicyclyl, heteroalicyclyl, benzyl 0449) wherein, 0450 n is an integer selected from about 2 to about 8: oraryl, wherein any ring in R is optionally substituted. 0451 X is a chemical- or photocrosslinking group 0433) each R" may be independently selected from tri Selected from the group consisting of tyl, MMT, and DMT: 0434 each R may be independently selected from 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-py ridyl, 2-nitrophenyl, 4-nitrophenyl, 3-carboxy-4-nitro phenyl, and 2,4-dinitrophenyl: OH, you. --ont. 0435 each J may be independently selected from Cl, Br, and I 043.6 L and Li may be independently selected from a COOH, --ol. -- c aiivil s 10437 “*” represents a portion of Land Lbound X' or N3, --to --on. Yi. US 2015/001 7246 A1 Jan. 15, 2015 26

-continued 0455 each of W° is independently selected from the group consisting of

COOH NO NO, NN

2

NO

xO NO and C 0456 each of W is independently selected from the group consisting of

10452 Y is a chemical- or photocrosslinking group O Selected from the group consisting of: SONa,

O X > F F F F CIHN W1 N \ N- No F 8. d F

O O F W3, ONH2, 0457 Preferably, the monomeric sugar alcohol may has x - the structure of a chemical formula selected from the group N N consisting of

O HO --ONH2,

O H HO sus 0453 W' is an independent linker selected from the r group consisting of —C(=O)—NH-, and, —NH-C O H O H (=O)—: HO Me On 1 - ON H2, 0454) each of J" is independently selected from Cl, Br Ns -W. and I; US 2015/001 7246 A1 Jan. 15, 2015 27

-continued -continued OH OH O W2 S ONH2 pi 3 sub O -W s n S 1 N- pi s O OH

to subuni.pi O OH H N N ONH2 and pi

O O O OH

W3No - usunpi 2

0460 Alternatively, the monomeric sugar alcohol has chemical formula selected from the group consisting of

0458 More preferably, the monomeric sugar alcohol has the structure of a chemical formula selected from the group consisting of

OH

insuls-N-pi O OH O

3 W&N pi O W3 O- s

NO Julu O- s O O OH H H N

N- Nan O O O O and OH

W3N1 S HN S a 1W2 S pi S 0461) Most preferably, the monomeric sugar alcohol has O O chemical formula selected from the group consisting of

0459 Alternatively, the monomeric sugar alcohol has OH chemical formula selected from the group consisting of subpi OH "Nuts-s-s 2 s US 2015/001 7246 A1 Jan. 15, 2015 28

-continued -continued OH OH O

W. I E w8, in-e-F2 F2 o1 O OH

FC N O OH N--in H I O O O Nullf NY1 OH O N, O / FC N E "N~ O OH O O OH W. l W3No 1sub2 N-4'-s- 2 and N, OH / - 2 F3 C N - Fl Sn-w: OH

2 W. I WS 1 N--F2 0463. The present invention also provides for a dimeric Sugar alcohol having the chemical structural Formula XVIII: N, f F3 C N (XXVIII) OH O O W. L3 L "Nuku'sFl x31 r W3 r Yy3 ORI,I OR'JI OR'J.I ORI 12 N, | FC N 0464) each of n1, m2, p 1, and p2 is independently OH selected from 0 and an integer selected from 1 to about W. 12; and n1+p1 is between 1 and 12, n2+p2 is between 2 Subpi and 12;

N 0465 r is 0 or 1; f I0466) wherein Wis selected from the group consisting N OF 3 FC N and of S - O - NH , NC-Calkyl-, -C(=O) OH NH-, -NHC(=O)— —S(=O)-, -S(—O), W. I —P(=S),O , and -P(=S)(=O)C)-. Jl N--t I0467 each of X is independently selected from -OH, -J, —C(=O)-CH-J, -OR. -S-S-R, NH O N. C(=O). CHCH-S-S-R, -S-C(=O)-CH / —C(=O)H, C(=O) R.-C(=O)CH, -C=C FC N R. N=N=N, -O-NH. O. NH-Fmoc, —O NH-Boc, O N-(Boc) —O N(-phthalim idyl), —C(=O)—NH-NH2, a phenol group, an 0462 Most preferably, the monomeric sugar alcohol has optionally substituted trifluoromethylphenyldiazirine, a chemical formula selected from the group consisting of diene, a dienophil, a 1.3-dipole, a dipolarophile, an alk ene, a ketene, an olefin, an alkene with allylic hydrogen, an optionally substituted N-hydroxysuccinimide ester, OH OH and aimidoester, a maleimide, and a phosphoramidite: I0468 each of Y is independently selected from the Nu'un - Nuhun's group consisting of-S-S-tButyl, - SR", S S - of o F2 R, -NH C(=O). CHCH S S R, NHR7, NH-Fmoc, NH-Boc, -O-NH. O. NH Nu'u. O -W', Fmoc, O NH-Boc, O N-(Boc) —O N(-phthal imidyl), an optionally substituted trifluoromethylphe US 2015/001 7246 A1 Jan. 15, 2015 29

nyldiazirine, al optionally substituted O NH S S R, NH-C(=O). CHCH N-hydroxySuccinimide ester, a imidoester, and a male S. S. R, an optionally substituted N-hydroxysuccimide imide; ester group, a optionally Substituted trifluoromethylphenyl 0469 each R" is independently selected from the group diazirine, and a maleimide group; wherein, consisting of hydrogen, C-Cs alkyl, benzoyl, acetyl, 0484) each R is independently selected from the group benzyl, C-C alkyl silyl cyclic ortho ester, actinide, consisting of 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, acetate, tetrahydropyranyl, tetrahydrofuranyl, and tet 5-nitro-4-pyridyl, 2-nitrophenyl, 4-nitrophenyl, 3-car rahydrothiofurany; boxy-4-nitrophenyl, and 2,4-dinitrophenyl. 0470 each Risindependently selected from hydrogen, 0485 The dimeric sugar alcohol compound may have X C-Cs alkyl, alicyclyl, heteroalicyclyl, benzyl or aryl, be an aminooxy group, Y be selected from the group con wherein any ring in R is optionally substituted; sisting of a maleimide group, a Sulfhydryl group, a disulfide 0471) each R is independently selected from a group group, a haloacetyl group, an azide group, and an alkyne consisting of benzoyl, acetyl, benzyl, C-C alkylsilyl, group. cyclic ortho ester, actinide, acetate, tetrahydropyranyl, 0486 The dimeric sugar alcohol compound may have X tetrahydrofuranyl, and tetrahydrothiofuranyl: be an optionally substituted N-hydroxysuccinimide ester, Y 0472 each R7 is independently selected from the group be selected from the group consisting of a maleimide group, consisting of trity1, MMT, and DMT: a sulfhydryl group, a disulfide group, a haloacetyl group, an 0473 each R is independently selected from the group azide group, and an alkyne group. consisting of 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 0487. The present invention also provides an embodiment 5-nitro-4-pyridyl, 2-nitrophenyl, 4-nitrophenyl, 3-car of a first Sugar alcohol-derived compound that has a chemical boxy-4-nitrophenyl, and 2,4-dinitrophenyl: formula 0474 each J is independently selected from the group consisting of Cl, Br, and I 0475 L and L is independently selected from the OH OH group consisting of a bond, —CH2—, —C(=O)— NH Cs aikvi 3, CH NH CO =O)—Cs X O Yl atty, , CH2 and C(=O)—NH-C-sit *, pi iii. p 0476 “*” represents a portion of Land Lbound X or Y3. 0488 wherein, 0477 Preferably, the dimeric sugar alcohol compound has 0489 n is an integer from 2 to about 8: a W selected from the group consisting of—S , —O , 0490 m is an integer from 1 to about 8: —S(=O)—, —S(=O) , —P(=S)O—, and - P(=S) (=O)C)-. 0491 p is an integer from 1 to about 2000; 0478. The dimeric sugar alcohol compound may have a 10492 each of X" is selected from the group consisting of: W which is —O— The dimeric sugar alcohol compound may have X is —C(=O) OH, r is 0, and Y is indepen dently selected from the group consisting of NH R", —NH-Fmoc, NH-Boc, -S-S-R, -S-S R7, SR 8, -NR18, yo" COR18, —S S-tButyl, —O NH-Fmoc. —O N-(Boc), and —O N(-phthalimidyl); wherein, O O O each R is independently selected from 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophenyl, 4-nitro phenyl, 3-carboxy-4-nitrophenyl, and 2,4-dinitrophenyl: r s' or each R is independently selected from the group consisting of trityl, MMT, and DMT, and when r is 1, then Y is OR' wherein; --co --out R" is independently selected from the group consisting of O O hydrogen, C-Cs alkyl, benzoyl, acetyl, benzyl, C-C alkyl silyl, cyclic ortho ester, actinide, acetate, tetrahydropyranyl. tetrahydrofuranyl, and tetrahydrothiofurany. --- - R18 0479. The dimeric sugar alcohol compound may have X O be a phosphoramidite when r is 0, Y is independently selected from the group consisting of —S—S—(C-C, alkyl)-OR7, S-S-R, -NH-TFA, -NH R", wherein WI 0480 each R" is independently selected from trityl, -- Nu y MMT, and DMT O 0481 each R is independently selected from 2-pyridyl, N 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitro M phenyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, and N S 2,4-dinitrophenyl. 0482. The dimeric sugar alcohol compound may have X CF3, Y s and Y not be the same. 0483. The dimeric sugar alcohol compound may have X and Y be independently selected from the group consisting of US 2015/001 7246 A1 Jan. 15, 2015 30

-continued -continued 2 W2 S H O W& S ry s N1S y s o W3 o s O1' O R18

CIHN O J. NH2, OH No ~ O x H - I r2

O o W3 N=C=S, NECEO and HY o1" R18

O NH2, OH --- x H - "? 0494 each W' is selected from the group consisting of N=C=S, NFCEO and C(=O) NH and NH C(=O)–: 0495 each J is selected from the group consisting of C1, Br or I: NH-NH2: 0496) R' is selected from the group consisting of hydro gen, Cs alkyl, alicyclyl, heteroalicyclyl, benzyl and aryl, wherein any ring in R' is optionally substituted each W is independently selected from the group consisting of 0493 each Y is selected from the group consisting of N NN O O O

s s s N 2 2 J| SR18 OR18 O O COOH NO ONH2, N3, ul x's R 18 NO2,2 n N s O 21 W1 \ Nu-N s NO ON O N N N 2 S N CF3, 1. s s > O H W2Ns1 S W2Ns1 S N- N NO and ON NO O

-- CIHN US 2015/001 7246 A1 Jan. 15, 2015 31 and, -continued 0497 each of Wis independently selected from the group OH OH O consisting of MeO O -W', pi iii. p O O O O OH OH SONa H

pi p

F F F F O O s

OH OH F and F. 2 HO pi O Sn-W, iii. p S

F F F OH OH MeO O S W2, 0498 Preferably, the first sugar alcohol-derived com pi Sn-Ns1 pound has a chemical formula selected from the group con sisting of:

OH OH OH OH H HO O N S W’ and HO O ONH2, pi in Y1 -n- ns1 pi iii. p O O OH OH OH OH H MeO O S W2 HO pi O p N~ e pi p r- n1S O O s O OH OH MeO O ONH2, 0499 More preferably, the first sugar alcohol-derived pi in Y1 compound has a chemical formula selected from the group consisting of:

HNO O ONH2, W& O W3, pi in Y1 2 No pi in Y1 o1

-- -- Xulu O ul s O O OH OH H H N N O N N \ Y1 2 in Yap O , and O O O

OH OH O S W2 s-/1 pi in Y1 ^- ns1” O O US 2015/001 7246 A1 Jan. 15, 2015 32

0500 Most preferably, the first sugar alcohol-derived -continued compound has a chemical formula selected from the group consisting of: OH OH O O W3 JI pi iii. p o1"

OH OH O W2 S O ONH2, O OH OH Ns 1 pi in Y1 OH OH W3 O S W2 No pi iii. p ns 1 s HNO O S.N.-W. O pi iii. p S OH OH O OH OH W2 S O ONH2 W&3 O Sn O pi iii. S, Ns 1. N- pi in Yap s p O OH OH O OH OH 2 3 H 2 WS -S O O -W HNO O N Sal-W, pi iii. p and pi in Yap r- S OH OH O O OH OH W2n1 S O 1.W3 S pi iii. p O HOOC O ONH2, O pi iii. p O OH OH H 0502. Alternatively, the first sugar alcohol-derived com N N O ONH2, pound has a chemical formula selected from the group con N- pi in Y1 sisting of: O O O OH OH OH OH HNO O NH N MeO O W pi iii. pi in Yap n p O O N, O OH OH I FC N O pi iii. p OH OH OH OH O HO O W pi iii. p N, | 0501 Alternatively, the first sugar alcohol-derived com FC N pound has a chemical formula has a chemical formula selected from the group consisting of: O OH OH W3 O W No pi iii. p O N, OH OH O f N O W3 FC pi iii. p o1" OH OH O O O O W O wi, O OH OH pi iii. p o1

W3n O N O pi it up ^- O O s US 2015/001 7246 A1 Jan. 15, 2015

-continued O OH OH OH OH H N O W N O ONH2, | NU/1 pi in Jp. pi iii. p O N. OH OH O r FC N O ONH2, O pi iii. p OH OH H OH O H O W O N N pi in J ^- s N N-' -W',3 N O O iii. p O A. N CF, OH O O 3 OH OH pi -W', OH J.in Y1 O WS2 -S O W. O pi iii. p OH OH H N O N pi p r O O s OH OH O OH OH H O N pi N p r W O N CF, O s OH OH OH OH O Sn -W HNO O W pi p S and pi iii. p OH OH

pi O p SN S -W.

OH OH 0504 Preferably, the first sugar alcohol-derived com pound has a p which is an integer from 1 to about 1000. 0505 Preferably, compound embodiment may be a sugar N alcohol derived compound wherein p is an integer from 1 to W about 500. N CF 0506 Optionally, the molecular weight of the first sugar OH OH alcohol-derived compound may be about 5 KDa to about 500 W O W KDa. Preferably, the molecular weight of the compound is about 10 KDa to about 100 KDa. More preferably, the N pi p O N and molecular weight of the compound is about 10 KDa to about N CF FC N 60 KDa. OH OH 0507 Optionally, the first sugar alcohol-derived com H pound is about 90% pure. Preferably, the compound is about N O W 95% pure. More preferably, the compound is about 97% pure. r pi iii. p O 0508. The molecular weight of the first sugar alcohol derived compound may be about 5KDato about 500 KDaand the compound may be about 90% pure. More preferably the molecular weight of compound may be about 10 KDa to about 100 KDa and the compound may be about 90% pure. 0503 Alternatively, the first sugar alcohol-derived com Most preferably, the molecular weight of the compound may pound has a chemical formula selected from the group con be about 10 KDa to about 60 KDa and the compound may be sisting of: about 90% pure. US 2015/001 7246 A1 Jan. 15, 2015 34

0509. The present invention also provides a second sugar (0512 Y is a chemical- or photocrosslinking group alcohol-derived compound having the chemical formula. selected from the group consisting of

OH O

pi WI Nu N No > wherein, O , s 0510 n is an integer from 2 to about 8: O 0511 X is a chemical- or photocrosslinking group -W', ONH2, selected from the group consisting of O

N

Ns 2 8. d W& S 1. S OH, OCH3, OCH2CH3, CF W1

2 H COOH, ONH2, Jl, WS-S N-ry O

N3. CHO, COCH, 0513 W' is an independent linker selected from the group consisting of C(=O) NH , and, NH C(=O)—: 0514 each J is independently selected from Cl, Brand I; W Nu-N \ s (0515 each W is independently selected from the group consisting of

O NM N NN N S 2 21 CF3, Y s N W O s s COOH NO

NO s H s n WS-S2 WS-52 N N 21 O CIHN H N No J1 ~ y O NO

O ON2 o W3 N H 3 o1 s 2 N

O s s NO and ON NO2: -NH2 and OH: N H US 2015/001 7246 A1 Jan. 15, 2015

0516 each W is independently selected from the group consisting of

O O SONa

O, O s F F F F --- and -- F F F

0517 Preferably the second sugar alcohol-derived com pound has a chemical formula selected from the group con sisting of 0519 Preferably the second sugar alcohol-derived com OH pound has a chemical formula selected from the group con sisting of resuls-N-pi OH "Nuhu ins -W, OH O O s W3 S Nu'ulo OH OH Ns1 N- 2: 2 O nus-s -W-, MeO pi ONH2, OH OH O to subunitspi O OH H N N ONH2 and pi

O O O OH O O s OH W3No - usunpi 2 sus- W2, pi ns1 0520 Preferably the second sugar alcohol-derived com OH pound has a chemical formula selected from the group con HO S W2 and sisting of pi r- ns1 8. O O OH O OH N Nu'uls W3 MeO S W2 N- pi o1 s e pi r- ns1 O O O OH O Nu'ul W3 0518. Preferably the second sugar alcohol-derived com r pi o1 s pound has a chemical formula selected from the group con O sisting of US 2015/001 7246 A1 Jan. 15, 2015 36

-continued -continued O OH OH W& - ul-In SS-W, "Null O pi r- S O N N and O OH W | W’ and N CF, FC N W3No 1suspi Ns 1 OH OH O H JI Nu'u'spi

S N- pi O O N. O l FC N 0521 Preferably the second sugar alcohol-derived com pound has a chemical formula selected from the group con 0522 Preferably the second sugar alcohol-derived com sisting of pound has a chemical formula selected from the group con sisting of

OH

subpi

N, | FC N OH OH E w8, resulpi - Nusus O N, OH f FC N pi O OH H N-is-in O O Nu'u's O NU/1 pi OH O N,

O f pi FC N O OH ------O O OH W3No -sulpi

N-4'-pi Sn S -W and N, f OH FC N E S W2. OH pi ns1

W3Ns 1 Nu'u'spi 0523 Optionally, the molecular weight of the second sugar alcohol-derived compound may be about 5 KDa to N, about 500 KDa. Preferably, the molecular weight of the com f pound is about 10 KDa to about 100 KDa. More preferably, FC N the molecular weight of the compound is about 10 KDa to OH about 60 KDa. 0524 Optionally, the second sugar alcohol-derived com Nu'u'.pi pound is about 90% pure. Preferably, the compound is about 95% pure. More preferably, the compound is about 97% pure. N, 0525 Preferably, the molecular weight of the second sugar f alcohol-derived compound may be about 5 KDa to about 500 FC N KDa and the compound may be about 90% pure. More pref erably the molecular weight of the compound may be about US 2015/001 7246 A1 Jan. 15, 2015 37

10 KDa to about 100 KDa and the compound may be about -continued 90% pure. Most preferably, the molecular weight of the com -- pound may be about 10 KDa to about 60 KDa and the com CIHN pound is about 90% pure. JI N s 0526. The present invention also provides a third sugar O O alcohol derived compound having a chemical formula O

o W3 o o1 OH OH R18 3 X sus, Y O S.-- iii. !p N1 NH2, OH H 0527 wherein 0528 n is an integer selected from 2 to about 8: N=C=S, N=C=O, 0529 m is an integer selected from 1 to about 8:

0530 p is an integer selected from 2 to about 2000; NH-NH2: 0531 wherein each W is independently selected from —S— —NH , —O— —NC-Calkyl-, -C(=O) 0533 a diene, a dienophil, a 1,3-dipole, a dipolarophile, an alkene, a ketene, an olefin, an alkene with allylic hydrogen, a benzophenone, an aryl diazonium, a vinyl (0532 each of X andY is independently a chemical- or Sulfone and an allyl Sulfone; photocrosslinking group selected from the group con 0534) L' and L is independently selected from a bond, sisting of —CH *, 0535 “*” represents a portion of L' and Lbound X or Y3. 0536) R' is hydrogen, Cs i? alicyclyl, heteroalicy SR, -NR18, yo". COR18, clyl, benzylor aryl, wherein any ring in R' is optionally Substituted. O O O 0537) each of W' is an independent linker selected from the group consisting of —C(=O)—NH —NH C se or (=O)—, 0538 each of J is independently selected from Cl, Br and I; --co --out Jl, 0539 each of W is independently selected from the O O group consisting of

--- x - R18 N NN O % 21 W N-, y COOH s NO2 NO, n N s O N M N S 2

CF, 1. NO > O W2 S W2 S 2 S s S s N O xc J. D US 2015/001 7246 A1 Jan. 15, 2015 38

-continued SA macromolecule is approximately 45% higher. The molecular and synthetic economy or efficiency can be advan tageous when making high MW biological conjugates as a single compound. (iii) Multiple accessible functional groups in SA macromolecules. In the drug delivery field, a polymer with variable MW compounds is usually used, such as poly (lactic-co-glycolic acid) (PLGA), which has been widely used in a host of FDA approved therapeutic devices. How (0540 each of W is independently selected from the ever, when PLGA is used for labeling or conjugating biomol group consisting of ecules, the functional groups are only limited to the termini, resulting in very low loading of the molecules. This is also true for PEG. SA macromolecules have functional groups O O throughout the molecules and are easily accessible. Depend ing on the architecture of the SA macromolecules, a high SONa density 3D network of drug can be achieved. (iv) Biodegrad ability and biocompatibility. SA macromolecules are based >{xO, O s on natural Sugar alcohols and can easily be degraded in vivo. F F F F Where labeling is via an OH group using the ester linkage, it can be degraded (hydrolyzed) in the presence of water. This property makes SA crosslinking reagents and SA macromol ecules great candidates for the delivery of small molecule drugs, as well as big molecule pharmaceuticals (e.g., biolog -R- and -- ics). F F F 0546. The present invention provides a collection of novel SA crosslinking reagents with various functional groups that 0541. Preferably, the third sugar alcohol derived com can be used for labeling, conjugating, and immobilizing mol pound embodiment has a p which is an integer selected from ecules of interest. In one aspect, the invention generally about 2 to about 1000. More preferably, p is an integer relates to a monomeric, linear, branched, or macrocyclic mul selected from 1 to about 1000. Most preferably p is an integer timer Sugar alcohol moiety comprising one or more of mono Selected from 1 to about 500. meric sugar alcohol unit B (a modified SA unit). When the 0542 Preferably, the third sugar alcohol derived com precursor compound comprises two or more monomeric pound has a molecular weight from about 5 KDa to about 500 Sugar alcohol units, each monomeric Sugar alcohol unit is KDa. More preferably, the molecular weight of the com bound to one or another monomeric Sugar alcohol unit pound is from about 10 KDa to about 100 KDa. Most prefer through a linking group W, wherein Wis formed by a reaction ably, the molecular weight of the compound is from about 10 between the X and Yportion of one monomeric unit with the KDa to about 60 KDa. XandY of any other monomeric unit. Each B" has the chemi 0543 Preferably, the third sugar alcohol derived com cal structure Formula XV: pound is about 90% pure. More preferably, the compound is about 95% pure. Most preferably, the compound is about 97% pure. (XV) 0544 Preferably, the molecular weight of the third sugar alcohol-derived compound may be about 5 KDa to about 500 KDa and the compound may be about 90% pure. More pref erably the molecular weight of compound may be about 10 KDa to about 100 KDa and the compound may be about 90% pure. Most preferably, the molecular weight of the compound may be about 10 KDa to about 60 KDa and the compound may be about 90% pure. 0545. The present invention provides methods and com positions, wherein a Sugar alcohol (SA) molecule is used as a wherein for each B", independently: backbone for building a series of novel chemical entities, 0547 each of n and p is independently selected from 0 and including SA crosslinking reagents and SA macromolecules, an integer selected from about 1 to about 12; and n+p is that are suitable for labeling and conjugating biomolecules between 1 and 12; and for drug delivery. The new molecules have significant 0548 r is 0 or 1: advantages over the existing system. (i) Super hydrophilicity 0549 each X, Y, and Z is independently selected from in which hydrophilicity is conferred from the hydrogen —OH, -J.-RJ, —C(=O)-J, —C(=O)—CH-J, NH bonds. The SA has hydrogen bond donors and acceptors C(=O)-CH-J, OR, OR,--OR. -O-Mesyl-O- available throughout the molecule, making the molecule Tosyl, -NH C(=O) CH-O-Mesyl, -NH C more similar to a network of water. As shown in FIG.1, SA is much more hydrophilic (lower Log P value, Log P=-3.995) than the comparable PEG linker (Log P=-2.117). (ii) Single and pure high MW SA macromolecules can be synthesized easily. For the same atom length, SA has a higher MW com pared to the PEG linker (FIG. 1). The MW of a similar length US 2015/001 7246 A1 Jan. 15, 2015 39

(-phthalimidyl), NH NH, C(=O) NH NH, -continued - NH C(=O) NH NH, NH CO-S). NH NH-toluenesulfonylhydrazide, R NH C (—NH)—NH, a benzophenone, an aryl diazonium, a dia Zoalkane, a diazoacetyl, an anthraquinone, a diazirine, an optionally substituted trifluoromethylphenyldiazirine, a diene, a dienophil, a 1,3-dipole, a dipolarophile, an alkene, a ketene, an olefin, an alkene with allylic hydrogen, a dicarbo , the nyl group, an epoxide, an oxirane, an organosilane, a phos phonium group, an ester, an anhydride, a carbonate group, a glyoxal, —C(=NH)—O R, a hydroxymethyl phosphine 0557. Each G' is independently selected from NR7, O, or derivative, an ethyl vinyl, a maleimide, a vinylsulfone, an S; each G’ is independently O or S; each G is independently allyl Sulfone, a thioester, a cisplatin derivative, an aziridine, selected from S. O. NR, or SO; each G' is independently O an acryloyl group. each R is independently selected from or NR; each R is independently selected from a bond, hydrogen, C-C alkyl, alicyclyl, heteroalicyclyl, benzyl or C-C alkyl, -(CH2CH2O)o , —(CH2CH2O)o aryl, wherein any ring in R is optionally substituted; CH , optionally substituted alicyclyl, heteroalicyclyl, aryl, 0550 each R is independently selected from benzoyl, a peptide, and a peptidomimetic oligomer, each R is a bond acetyl, benzyl, C-Cs alkylsilyl, tetrahydropyranyl, tetrahy or —CH2—, each R is independently selected from hydro drofuranyl, and tetrahydrothiofuranyl: gen, C-C alkyl, —(OCH2CH), optionally substituted 0551 each R7 is independently selected from trityl, MMT, alicyclyl, and optionally Substituted heteroalicyclyl; and each and DMT: R is independently C-Cs alkyl. 0552 each R is independently selected from 2-pyridyl, 0558. In some preferred embodiments, linking group W is 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophe selected from —S— —O— —NH-, -NC-C alkyl-, nyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, and 2,4-dinitro - C(=O)NH-, -NHC(=O) , S(=O) , S(=O) phenyl: — —P(=S)O—, or—P(=S)(=O)C)—. 0553 each J is independently selected from Cl, Brand I. 0559. In certain more preferred embodiments, W is 0554 each of L', L, and L is independently a R or selected from —S— —O— —S(=O)— —S(=O) , R V R2 * —P(=S)O—, or—P(=S)(=O)C)—. In certain even more 0555. In some embodiments the linker is R V R preferred embodiments, W is —O—. * , wherein “” represents a portion of the linker bound to X, 0560. In some embodiments a “modified” SA unit refers to Y, or Z, and V is a linking group. a SA unit in which one or more of its OH groups have been 0556. In some embodiments each V and W is indepen chemically modified by substitution with another functional dently selected from a Diels-Alder adduct, a 1,3-dipolar group. In this case, L' connecting to an OH substituted X is adduct, —C(=G')-G-, -G-C(=G')-, -G-, -G-C(=G')-G-, —CH ; L connecting to an OH substitutedY is —CH : and any L connecting to an OH substituted Z is a bond. In some embodiments, a “modified SA unit refers to a SA unit in which one or both of its —CH-OH groups has been oxi CH=N. NH C(O) , CH-NH NH-C(O) , dized, usually to an aldehyde or carboxylic acid, and then further modified with other functional groups. In this case, L' and L are bonds. In some embodiments, a “modified” SA unit also refers to a SA unit in which the hydrogenatomatone or more of the OH groups has been replaced by a chemical protecting group, leaving group, or other functional group. In this case, each of L' and L is —CH2— and L is a bond. Preferably, each-L-Z is —OR' wherein each R" is indepen dently selected from the group consisting of hydrogen, C-Cs alkyl, benzoyl, acetyl, benzyl, C-C alkylsilyl cyclic ortho ester, actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl. and tetrahydrothiofurany. The following is a list of various simplified structures of an SA B' unit.

3-7. (XXXI) his O US 2015/001 7246 A1 Jan. 15, 2015 40

-continued hydrazines capable of reacting with aldehyde or ketone

(XXXII) groups to form the hydrazine linkage. In another embodi ment, the functional groups X, Y, and Zare semi or thiosemi carbazides capable of reacting with aldehyde or ketone groups to form the semi or thiosemicarbazone linkage. In another embodiment, X or Y can be amine functional groups. Amine groups can react with aldehydes through Schiffbase formation and the formed carbon amine double bond can be (XXXIII) further reduced to a stable secondary or tertiary amine bond. Amine groups can also react with an active hydrogen-con taining compound in the presence of formaldehyde (Mannich reaction). 0566 In some embodiments the functional groups X, Y, and Z are able to react with certain reactive (or replaceable) hydrogens that exist in certain biomolecules. For example, X. (XXXIV) Y, or Z may be a diazonium group that reacts with active L L& x1 Y hydrogen sites on aromatic rings to produce covalent diazo bonds. OR3 0567. In some preferred embodiments the functional n-p groups X, Y, and Z are photoreactive groups that can be induced to couple with molecules of interest by exposure to 0561. In certain embodiments, the functional groups X,Y, UV light. The preferred photoreactive groups include, for and Z are amine reactive groups. The primary coupling reac example, diazirine groups. More preferable than a diazirine tion for amine is through acylation or alkylation. The pre group is 3-trifluoromethyl-3-aryaldiazirine. Other preferred ferred amine active groups include, for example, carboxylic photoreactive groups are aryl azides, halogenated aryl azides, acid, ketenes, isothiocyanate, isocyanate, acyl azides, acyl benzophenones, anthraquinones, diazos Such as diazotrifluo halides, N-hydroxysuccinimide (NHS) ester, sulfonyl chlo ropropionates and diaZopyruvate, and psoralen derivatives. ride, carbonyl groups such as aldehydes, ketones, and glyox 0568. In some preferred embodiments the functional als, epoxides or oxiranes, carbonate groups, aryl halides such groups X, Y, and Z are dienes, dienophile groups, or alkenes as fluorobenzene derivatives, alkyl halides, imidoester, or that are capable of linking molecules through the Diels-Alder imidate functional groups, anhydrides, fluorophenyl ester, reaction. Preferably, X, Y, or Z may be an azido functional and hydroxymethyl phosphine derivatives. In a particular group or alkyne that is capable of linking molecules through example, XorY is an arginine-reactive group. The guanidinyl 3+2] cycloaddition. In another embodiment X, Y, or Z may group on the arginine side chain can be specifically targeted be an alkene with an allylic hydrogen (the ene) or a multiple using 1,2-dicarbonyl reagents, such as the diketone group of bond (the enophile). In another embodiment X,Y, or Z may be glyoxal. 1,3-dipole or a dipolarophile (substitute alkenes). 0562. In certain embodiments the functional groups X, Y, 0569. In some preferred embodiments X is a protected and Zarethiol reactive groups. The primary coupling reaction amine and Y is an acid wherein each -L-Z is an —OR". SA for thiol is through alkylation or disulfide exchange. The crosslinking reagents can be incorporated into any molecule preferred thiol reactive groups include, for example, thiol, (e.g., peptide and nucleic acid oligomers) through the solid haloacetyl and alkyl halides, maleimide, aziridines, acryloyl phase synthesis strategy. For example, X can be a fluorenym derivatives, arylating agents such as benzene derivatives that ethyloxycarbonyl (Fmoc) protected amine. In another possess either halogen or Sulfonate groups on the ring, thiol example, X can be a tert-butyloxycarbonyl (Boc) protected disulfide exchange reagents such as pyridyl dithiol and thi amine. In another example, X can be a triphenylmethyl chlo olnitrobenzoic acid, vinylsulfone, cysteine derivatives, ride (trity1), dimethoxytrityl (DMT), or monomethoxytrityl thioesters, and cisplatin derivatives. (MMT) protected amine, so SA crosslinking reagents can be 0563. In certain embodiments the functional groups X, Y, incorporated into an oligomer through solid phase synthesis. and Z are carboxylic acid reactive groups. The preferred 0570. In some preferred embodiments X is a protected carboxylic acid reactive groups include, for example, OH, thiol (e.g., trity1 or t-butyl thiol protected) and Y is an acid amines, thiols, diazomethane and diazoacetate derivatives, wherein each-L-Z is an—OR, wherein R is independently and acylimidazole leaving groups. selected from hydrogen, C-C alkyl, -(OCH2CH), 0564. In certain embodiments the functional groups X, Y, optionally substituted alicyclyl, and optionally substituted and Z are OH reactive groups. The preferred hydroxyl reac heteroalicyclyl. tive groups include, for example, epoxides or oxiranes, alkyl 0571. In some preferred embodiments X is a protected halogens, carboxylic acid and its active esters, isocyanate, amine (e.g., trity1, DMT, or MMT protected) and Y is a isothiocyanate, phosphonium intermediates, tosylate or phosphoramidite wherein each -L-Z is an —OR. mesylate, Sulfonyl chlorides, anhydrides, acyl azides, tet 0572. In some embodiments the X and Y groups are the rahydropyranyl, tetrahydrofuranyl, tetrahydro thiofuranyl, same, wherein each -L-Z is an —OR' and as such a homo ethyl vinyl, trityl halides, fluorobenzene derivatives, silyl bifunctional SA functional reagent. Examples of homobi halides, and ketenes. functional SA functional reagents include homobifunctional 0565. In some preferred embodiments the functional NHS ester, homobifunctional imidoester, homobifunctional groups X, Y, and Z are aminooxy groups capable of reacting dithiopyridyl group containing crosslinking reagent, homo with aldehyde or ketone groups to form the oxime bond. In bifunctional maleimide group containing crosslinking another embodiment, the functional groups X, Y, and Z are reagent, homobifunctional alkyl halide containing crosslink US 2015/001 7246 A1 Jan. 15, 2015

ing reagent, homobifunctional photoreactive crosslinking 0579 each of n and p is independently selected from 0 and reagent, homobifunctional aldehyde or ketone, homobifunc an integer selected from 1 to about 12; and n+p is between 2 tional epoxide, homobifunctional hydrazide, homobifunc and 12; ris 0 or 1: Land L'is independently selected from a tional aminooxy, and homobifunctional diazonium. bond, —CH2 *, —C(=O)—NH-Cls if *, —CH2 0573. In some embodiments the X and Y groups are dif NH C(=O)—Cs aikvi *, —CH C(=O) NH Cs ferent wherein each -L-Z is an —OR" and as such a hetero aii-, “*” represents a portion of Land L'bound X orY. bifunctional SA crosslinking reagent. In certain preferred Each R" is independently selected from the group consisting embodiments X is an amine-reactive group and Y is a sulfhy of hydrogen, C-Cs alkyl, benzoyl, acetyl, benzyl, C-Cs dryl-reactive group. In certain preferred embodiments X is a alkylsilyl cyclic ortho ester, actinide, acetate, tetrahydropy carbonyl-reactive (aldehyde/keto). Such as aminooxy, hydra ranyl, tetrahydrofuranyl, and tetrahydrothiofurany. Zine, semicarbazide, or thiosemicarbazide, and Y is a sulfhy dryl-reactive group. In certain preferred embodiments X is an (0580. In certain embodiments each of X" is independently amine-reactive group and Y is a photoreactive group. In cer selected from -OH, -J, —C(=O) CH-J, —OR, tain preferred embodiments X is a sulfhydryl-reactive group S S R, NH-C(=O). CHCH S S R, and Y is a photoreactive group. In certain preferred embodi —S—C(=O)—CH, - C(=O)H, —C(=O) R, ments X is a carbonyl-reactive group and Y is a photoreactive C(=O)CH, -C=C R, N=R"—N, O NH, group. In certain embodiments X is a carboxylate-reactive —O NH-Fmoc, —O NH-Boc, —O N-(Boc) —O N group and Y is a photoreactive group. In certain embodiments (-phthalimidyl), —C(=O)—NH-NH2, a phenol group, an X is a ketone or aldehyde and Y is a photoreactive group. optionally substituted trifluoromethylphenyldiazirine, a 0574. In addition, SA crosslinking reagents may incorpo diene, a dienophil, a 1,3-dipole, a dipolarophile, an alkene, a rate the cleavable linkage that allows systematic drug release ketene, an olefin, an alkene with allylic hydrogen, an option for a conjugate in vivo. For example, aminooxy functional ally substituted N-hydroxysuccinimide ester, a imidoester, a groups may be introduced at the termini of the crosslinking maleimide, a phosphoramidite; each R is independently reagent, and a cleavable oxime linker may be used to release selected from hydrogen, C-C alkyl, alicyclyl, heteroalicy the molecules being crosslinked. In another example, disul clyl, benzyl or aryl, wherein any ring in R is optionally fide bonds may be incorporated, allowing the release of the substituted; each Risindependently selected from 2-pyridyl, molecules in a reductive environment. Moreover, SA 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophe crosslinking reagents can link two or more SAS through nyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, or 2,4-dinitro another bond that may be enzymatically cleaved in vivo. phenyl; and each J is independently selected from Cl, Br, or I. 0575. Furthermore, SA crosslinking reagents having two 0581. In some preferred embodiments X is C(=O)— or more OH groups (secondary OH groups) in the backbone OH, r is 0, and Y is independently selected from NH R7. may themselves be cleavable linkages. For instance, an SA NH-Fmoc, NH-Boc, -S-S-R, -S-S R7, backbone with two or more OH groups may be cleaved by —S S-tButyl, - O NH-Fmoc, —O N-(Boc), and periodate oxidation to generate a new aldehyde functional —O N(-phthalimidyl); wherein; each R is independently group. selected from 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro 0576 Furthermore, SA crosslinking reagents may incor 4-pyridyl, 2-nitrophenyl, 4-nitrophenyl, 3-carboxy-4-nitro porate a reducing Sugar unit in the backbone. In at least one of phenyl, and 2,4-dinitrophenyl; each R is independently SA or SA, r is 1. For example, if one of the reducing Sugar selected from trityl, MMT, and DMT, and when ris 1, then Y' units is the reduced form of fructose, an SA crosslinking is OR', wherein; each R" is independently selected from the reagent containing trifunctional group X,Y, and ketone can be group consisting of hydrogen, C-C alkyl, benzoyl, acetyl, envisioned. In some embodiments X may be a protected benzyl, C-C alkylsilyl cyclic ortho ester, actinide, acetate, amine and Y an acid. This configuration is useful for intro tetrahydropyranyl, tetrahydrofuranyl, and tetrahydrothio ducing a ketone functional group into a peptide or oligo furany. through solid phase synthesis. This ketone handle can be used conveniently for the conjugation of other molecules. 0582. In some preferred embodiments X is a phosphora midite; when r is 0, Y is independently selected from 0577 Monomeric SA and Linear SA Crosslinking -S-S (C-C alkyl)-OR', S-S-R, NH-TFA, or Reagents NH R", wherein each R7 is independently selected from 0578. In one aspect, the present invention provides a trityl, MMT, or DMT and each R is independently selected monofunctional, homobifunctional, and heterobifunctional from 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-py crosslinking reagent comprising one SA unit. The compound ridyl, 2-nitrophenyl, 4-nitrophenyl, 3-carboxy-4-nitrophe has the chemical structure Formula XXVI: nyl, or 2,4-dinitrophenyl. 0583. In some preferred embodiments X and Y are not the same.

(XXXVI) (0584) In some preferred embodiments one of each X and Y is O NH, S S R NH CO-O) CHCH S S R, an optionally substituted N-hydrox ySuccimide ester group, a optionally Substituted trifluorom ethylphenyldiazirine, or a maleimide group, wherein each R is independently selected from 2-pyridyl, 4-pyridyl, 5-nitro 2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophenyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, or 2,4-dinitrophenyl. US 2015/001 7246 A1 Jan. 15, 2015 42

0585. In some more preferred embodiments the mono -continued meric Sugar alcohol has the following structure: 8. d W2 CF Ns -S W Nuespi W3 S 0586 Wherein n is an integer selected from about 2 to Ns1 N about 8: O 0587 each of X is a chemical-orphotocrosslinking group selected from the groups consisting of: {-OH, -—OCHs. —{OCH2CH, -COOH, - ONH2, — J. —{ N. (0588 each of W' is an independent linker selected from ——CHO, — COCH, the group consisting of —C(=O) NH and —NH C (=O)—:

O 0589 each of J is independently selected from Cl, Br or I: 0590 each of Wis independently selected from the group consisting of W1 N-1 N \

O , N NN N V N 4. 2

COOH NO "> NO, NN > s O1 , W2S-X S W2s.r-ry S 2 s O s CIHN NO No r y ON s O s CN NO2 and ON, NO 0591) each of Wis independently selected from the group each ofY is a chemical- or photocrosslinking group selected consisting of from the group consisting of O O SONa \ CIHN N > O, > O s wi1N1 N F F F F O O W3 -R- and F x - --on F F F US 2015/001 7246 A1 Jan. 15, 2015 43

0592. In some preferred embodiments the monomeric -continued Sugar alcohol compound is a monofunctionalized Sugar alco OH hol crosslinking reagent with the following chemical struc- WS-S sué-IN S-N-W tures: S N-- pi -n- S O

OH resuls-N- 0594. In some preferred embodiments the monomeric pi O Sugar alcohol compound is a heterobifunctional aminooxy OH crosslinking reagent with the following chemical structures: resul- H NC r- OH O O 2 OH OHs HNO2 N-- S ns1W4, 2 OH "N-4'-'s -W , MeO pi ONH2, 2 H

W& S 1 S N- N pi ONH2,

O O OH OH H HOOC ONH2, MeO N N pi

pi r- O OH O O s H OH N N ONH2 and pi

sus-pi S Ns1W2 s O N O OH H O OH HO N S W2 and

pi r- Ns1 W& O pi ONH2. O 0595. In some preferred embodiments the monomeric sus-pi r- Sn-W.S Sugar alcohol compound is a heterobifunctional N-hydrox ySuccimide ester crosslinking reagent with the following O chemical structures

0593. In some preferred embodiments the monomeric O Sugar alcohol compound is a homobifunctional crosslinking reagent with the following chemical structures OH O N NNu'u. N-1 No1 w8, OH O O HNO ONH2, pi OH O O OH O H N wi, W3 wi, JI pi o1 No pi o1 -- O

Xulu 3 O OH 2 ONo pi o1. O "s-l-el-in Sn-w s OH N O US 2015/001 7246 A1 Jan. 15, 2015 44

-continued -continued OH O OH H H W2Ns1 S N- Nuebuspi o1W3. r N--pi O O N. / 0596. In some preferred embodiments the monomeric FC N Sugar alcohol compound is a photocrosslinker reagent with the following chemical structures 0597. In some preferred embodiments the monomeric Sugar alcohol compound is a heterobifunctional crosslinking reagent with the following chemical structures OH

subpi

O

"sub-pi -sul W3 s pi O 1. O / OH FC N O N HN N OH pi r- s N --- O O n- pi OH O O O f S HN N FC . pi r- s O O

WS-2 N-- 0598. In one aspect, the present invention provides a dimeric sugar alcohol with the chemical structure Formula N, XVIII: / FC N OH (XXVIII)

Null-pi O O 5 L N x31 L * W3 * Ny3 / ORI ORI ORI ORI FC N pal pl n2 p2 0599 each of n1, m2, p 1, and p2 is independently selected sub- from 0 and an integer selected from 1 to about 12; and n1+p2 pi is between 1 and 12, n2+p2 is between 2 and 12: N and 0600 r is 0 or 1: N / 8. 0601 wherein W3 is selected from —S —O—, N - NH-, -NC-Calkyl-, - C(=O)NH-, -NHC N CH FC (=O)— —S(=O)— —S(=O) , —P(=S)O—, or US 2015/001 7246 A1 Jan. 15, 2015

0602 each of X is independently selected from -OH,-J, 2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophenyl, 4-nitrophenyl, —C(=O)—CH-J, OR, S S R NH C 3-carboxy-4-nitrophenyl, and 2,4-dinitrophenyl. (=O). CHCH-S-S R. —S—C(=O)—CH, I0614. In some more preferred embodiments X is an ami —C(=O)H, -C(=O) R. C(=O)CH, -C=C R, nooxy group and Y is selected from the group consisting of - N=N*—N, - O NH, - O NH-Fmoc, O. NH a maleimide group, a Sulfhydryl group, a disulfide group, a Boc, —O N-(Boc) —O N(-phthalimidyl), —C(=O)— haloacetyl group, an azide group, and an alkyne group. In NH NH, a phenol group, an optionally substituted trifluo some embodiments X is an optionally substituted N-hydrox romethylphenyldiazirine, a diene, a dienophil, a 1,3-dipole, a ySuccinimide ester, Y is selected from the group consisting dipolarophile, an alkene, a ketene, an olefin, an alkene with of a maleimide group, a sulfhydryl group, a disulfide group, a allylic hydrogen, an optionally Substituted N-hydroxysuccin haloacetyl group, an azide group, and an alkyne group. imide ester, a imidoester, a maleimide, a phosphoramidite; 0615. In another aspect, the sugar alcohol is a linear trimer 0603 each ofY is independently selected from —S S or a higher oligomer having the following structure tEutyl, - SR", -S-S-R, -NH C(=O)-CHCH S–S R, NHR'. - NH-Fmoc, NH-Boc, O NH, (XX)

—O NH-Fmoc, —O NH-Boc, —O N-(Boc) —O N (-phthalimidyl), an optionally substituted trifluorometh ylphenyldiazirine, an optionally substituted N-hydroxysuc cinimide ester, a imidoester, a maleimide; 0604 each R is independently selected from benzoyl, acetyl, benzyl, C-Cs alkylsilyl, tetrahydropyranyl, tetrahy drofuranyl, and tetrahydrothiofuranyl: 0605 each R" is independently selected from trity1, MMT, and DMT: 0606 each R is independently selected from 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophe 0616 wherein V is an integer selected from 2 to 2000. nyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, and 2,4-dinitro I0617. In some preferred embodiments-L-Z is —OR" and phenyl: has the following structure XXI 0607 each J is independently selected from Cl, Br, and I 0608 L and L is independently selected from a bond, CH2 *, —C(=O)—NH-C-src *, —CH2 NH (XXI) CO) C1-8 alkyl 3, CH, C(=O) NH Ci-saiki O L2 L 2 0609 “*” represents a portion of Land Lbound XorY. w1. * L 0610. In some embodiments W is selected from the group Y consisting of —S— —O— —S(=O)— —S(=O) , ORI ORI —P(=S),O , or - P(=S)(=O)C-. More preferably, W pi p a is —O—. 0611. In some embodiments, when X is C(=O) OH: r is 0, and Y is independently selected from NH-R7. -NH-Fmoc, NH-Boc, -S-S-R, -S-S R7, —S S-tButyl, - O NH-Fmoc, —O N-(Boc), and wherein R' is independently selected from the group consist —O N(-phthalimidyl); wherein; each R is independently ing of hydrogen, C-Cs alkyl, benzoyl, acetyl, benzyl, C-Cs selected from 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro alkylsilyl cyclic ortho ester, actinide, acetate, tetrahydropy 4-pyridyl, 2-nitrophenyl, 4-nitrophenyl, 3-carboxy-4-nitro ranyl, tetrahydrofuranyl, and tetrahydrothiofurany. phenyl, and 2,4-dinitrophenyl; each R is independently 0618. In some more preferred embodiments wherein each selected from trity1, MMT, and DMT, and when ris 1, then Y r is 0 and has the following structures: is OR', wherein; R' is independently selected from the group consisting of hydrogen, C-C alkyl, benzoyl, acetyl, benzyl, C-Cs alkylsilyl cyclic ortho ester, actinide, acetate, tetrahy (XXII) L L2 L L2 dropyranyl, tetrahydrofuranyl, and tetrahydrothiofurany 1 1 0612. In some embodiments, when X is a phosphoramid X W Y, ite; When ris 0, Y is independently selected from S S - ORI ORI (C-C alkyl)-OR7, S-S-R, NH-TFA, NH-R7. n-p n-p wherein each R" is independently selected from trityl, MMT, and DMT; each R is independently selected from 2-pyridyl, 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitrophe nyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, and 2,4-dinitro phenyl. (XXIII) L L2 0613. In some preferred embodiments, at least one of each 1 X and Y is O. NH S S R, NH C(=O) X W Y CHCH S S R, an optionally substituted N-hydrox ORI ORI ySuccimide ester group, an optionally Substituted trifluorom n-p nilp ethylphenyldiazirine, or a maleimide group; wherein each R is independently selected from 2-pyridyl, 4-pyridyl, 5-nitro US 2015/001 7246 A1 Jan. 15, 2015 46

0619. In another aspect, the sugar alcohol-derived com- -continued pound has the structure -NH2, OH N OH OH x.

N=C=S, NECEO and

Wherein: NH-NH2: 0620 n is an integer selected from 2 to about 8: 0621 m is an integer selected from 1 to about 8: (0624) each Y is a chemical- or photocrosslinking group 0622 p is an integer selected from about 1 to about 2000; selected from the group consisting of: 0623) each of X" is a chemical-orphotocrosslinking group selected from the group consisting of: O O O -r -NR, yo" --tor J SR 18 OR 18 O O O ONH2, N3, ul

J| SR18 OR18 O

COOH, ONH2, Jl, WI \ Nu-N s

O H W2 S W2 S N O Ns 1. Ns 1. o W3 O 18 o1 -- R H CIHN N O

No r O N -NH2, OH H

o W3 o s o1 s N=C=S, NECEO and R18 US 2015/001 7246 A1 Jan. 15, 2015 47

-continued -continued F F F F NH-NH

------F F F 0625 each of W' is an independent linker selected from the group consisting of —C(=O)—NH and —NH C (=O)—: 0630. In some embodiments only one of the OH groups is 0626) each of J is independently selected from Cl, Br or I: modified with a crosslinking group and the linear Sugar alco 0627) R' is hydrogen, Cls talicyclyl, heteroalicyclyl, hol-derived compound has the following chemical structures: benzyl, or aryl, wherein any ring in R' is optionally substi tuted; OH OH (0628 each of Wis independently selected from the group consisting of in-ul-J-pi iii. p ONH2, O OH OH N NN H HO O N NC || 4. 2 pi iii. p O O OH OH COOH NO NO Mosul,pi usu,iii. Jp n N OH OH O 21 3 unusuu,pi iii. J-p O -W', O OH OH H

NO MeO pi O iii. p Nr- s ON O O N OH OH 2 2 s N pi iii. S resulus-J->p -W,

NO ON NO

OH OH 2 and subursuspi iii. J-Sp S -W s OH OH 0629 each of Wis independently selected from the group H 2 consisting of HO pi O iii. p N~'s S -W and O OH OH O O S W2 pi iii. p S s SONa, MeO O r- n1 O XKO x O 0631. In some embodiments two of the primary OH groups are modified with the same crosslinking groups, a homobifunctional crosslinker with the following chemical Structures: US 2015/001 7246 A1 Jan. 15, 2015

OH OH O OH OH O HNO O ONH2, W& O -W, pi iii. O pi iii. O p p -- CIHN OH OH

O s No pi iii. o1 O O OH OH H H N N O N N pi iii. s and p O O O O OH OH H H N O N

W2 / S p S NS O O

In some embodiments two of the primary OH groups are -continued modified with different crosslinking groups, an aminooxy O OH OH heterobifunctional crosslinker with the following chemical W3 O ONH2, and Structures: s------p OH OH O

OH OH 3 pi iii. O WS2 1S O ONH2, to-bus- p -W. pi iii. p OH OH In some embodiments two of the primary OH groups are 2 modified with different crosslinking groups, an active ester HNO O SN-W, pi iii. S heterobifunctional crosslinker with the following chemical p Structures: OH OH W2 S O ONH n1 2: O S pi iii. p OH OH O O N O W3 OH OH pi iii. o1 p H S W2 HNO O N n1' O O pi iii. S p O O O OH OH OH OH W& O N O pi iii. s HOOC O ONH2, p pi iii. p O O O OH OH O OH OH O W3 H JI pi iii. o1 N N O ONH2, p pi iii. p O O OH OH O O O W& O SS-W. O pi iii. S OH OH p O HNO O NH N O OH OH pi iii. s p W3 O S O O s------p Ns, US 2015/001 7246 A1 Jan. 15, 2015 49

-continued -continued OH OH O OH OH W& -S O -W and 2 S pi iii. O "Sull-les-J-pi iii. p Sn-W,S p OH OH - O N

W& -S O -W \N CF3 p S n-r pi iii. O OH OH O

"Null-les-J-W.pi iii. p 0632. In some embodiments the linear SA molecule is a photocrosslinking reagent with the following chemical struc N, tures: FC N OH OH OH OH W O ONH2, pi iii. p Mosul,pi usulin Y N N, f N CF3 FC N OH OH OH OH wusuous--"pi iii. pi iii. p N and p N, \ CF FC3 N/ | N FC N OH OH O OH OH H N Null-les-J-" W3 O 1suhunupi in n-,J r pi in-, O N, ? FC N OH OH O 0633. In some embodiments the linear SA molecule has the following chemical structures:

N \ OH OH N CF O N O ONH2, OH OH pi iii. H p OH OH N n-n Nu'ulou.J-pi in n1 O O N, S O ONH2, pi iii. FC | p N OH OH O O OH OH H N O w8, W. O N N -)- -busp pi in Y1 N-n- OH OH O N O O E O w8, \ CF N 3 ------p O OH OH H N O N N pi iii. s p O O US 2015/001 7246 A1 Jan. 15, 2015 50

-continued 0638. In some embodiments X orY functional groups can O be attached to the SA backbone through extra linkers (L' and OH OH L). FIG.3 illustrates an embodiment of a general approach H E O N N for synthesizing monofunctional, homobifunctional, and het pi iii. s erobifunctional SA crosslinking reagents with extra linkers. p The method consists of: (i) providing a Sugar alcohol inter O O mediate (I4) with functional group X at one termini and OH OH protected primary OH group at the other termini and (ii) combining the SA with reagents that react with X to form an N O S W2 and intermediate (I10) with an extra linker (L). In some embodi -)- ---p Ns 1 ments the linker is a stable linker. In some embodiments the OH OH linker is a cleavable linker. In some embodiments the linker is S O S W2. a reversible linker. ------p ns1 0639. In one approach, I10 can be fully deprotected to obtain a monohydroxyl SA crosslinking reagent with an extra linker (P8). Further alkylation provides an SA crosslinking reagent containing a single active functional group (P9). Methods of Synthesizing Linear SA Crosslinking Reagents Alkylation can also be achieved in the beginning by blocking the secondary OH groups with alkyl groups. 0634. The present invention provides methods for synthe sizing SA crosslinking reagents, for example, a method for 0640. In one approach, I10 can be combined with reagents synthesizing monodisperse, pre-determined high MW SA that selectively deprotect the primary OH group to generate I11, which can further react with reagents that introduce macromolecules. FIG. 2 illustrates an embodiment of agen another functional group X that is not an OH group. Depro eral approach for synthesizing monofunctional, homobifunc tection of the secondary OH groups of I12 generates a het tional, and heterobifunctional SA crosslinking reagents. The erobifunctional SA crosslinking reagent with an extra linker method consists of: (i) providing a Sugar alcohol; (ii) com (P13). Alkylation of the secondary OH groups results in P14, bining the SA with reagents that can selectively protect the an alkylated heterobifunctional SA crosslinking reagent with two terminal primary OH groups to forman intermediate (I1); an extra linker. Alkylation can also be achieved in the begin (iii) combining the SA with reagents that react with the sec ning by blocking the secondary OH groups with alkyl groups. ondary OH to form an intermediate (I2); (iv) combining the In another case, direct alkylation of the primary OH group, SA with reagents that selectively deprotect one of the primary followed by deprotection of the secondary OH groups, gen OH groups to generate an intermediate (I3); and (V) reacting erates a monoalkylated monosubstituted SA crosslinking with reagents that introduce different functional groups X reagent with an extra linker (P10). other than OH groups (I4). 0641. In another approach, I12 can further react with 0635. In one approach, I4 can be fully deprotected to reagents to generate a second linker with a functional group obtain a monosubstituted SA crosslinking reagent (P3). Fur (I14). Deprotection of the secondary OH groups generates a ther alkylation of the monosubstituted SA crosslinking heterobifunctional SA crosslinking reagent with two extra reagent P3 generates an SA crosslinking reagent with a single linkers (P11). Alkylation of the secondary OH groups results active functional group (P4), an alkylated monosubstituted in P12, an alkylated heterobifunctional SA crosslinking SA crosslinking reagent. Alkylation can also be achieved in reagent with two extra linkers. Alkylation can also be the beginning by blocking the secondary OH groups with achieved in the beginning by blocking the secondary OH alkyl groups. groups with alkyl groups. 0636. In one approach, I4 can be combined with reagents 0642. In one approach, homobifunctional SA crosslinking that selectively deprotect the primary OH group to generate reagent I9 can react with reagents that generate linkers at both I5, which can further react with reagents that introduce non termini (I15). Deprotecting the secondary OH groups of I15 hydroxyl functional group Y. Deprotection of the secondary generates a homobifunctional SA crosslinking reagent with OH groups of I6 generates a heterobifunctional SA crosslink two extra linkers (P15). Alkylation of the secondary OH ing reagent, P1. Alkylation of the secondary OH groups groups results in P16, an alkylated homobifunctional SA results in P2, an alkylated heterobifunctional SA crosslinking crosslinking reagent with two extra linkers. Alkylation can reagent. Alkylation can also be achieved in the beginning by also be achieved in the beginning by blocking the secondary blocking the secondary OH groups with alkyl groups. In OH groups with alkyl groups. another approach, direct alkylation of the primary OH group, 0643. The methods described herein utilize the general followed by the deprotection of secondary OH groups gener protection and deprotection mechanism for OH groups. The ates a monoalkylated monofunctional SA crosslinking orthogonal OH protecting groups (PG1, PG2, PG3) can be reagent (P5). selected from the varieties of protecting groups (for example, 0637. In one approach, I2 can be combined with reagents as disclosed in Greene, TW: Wuts, PGM Protective groups in that deprotect both primary OH groups to generate I8, which organic synthesis, third edition, John Wiley & Sons, Inc. can further react with reagents to introduce functional groups 1999). The techniques and experiments involved in the actual at both termini. Deprotection of the secondary OH groups of reduction in practice involve all classical organic synthesis I9 generates a homobifunctional SA crosslinking reagent methods and can be easily implemented by a skilled artisan in (P6). Alkylation of the secondary OH groups results in P7, an view of the disclosure herein. FIGS. 4 and 5 provide numer alkylated homobifunctional SA crosslinking reagent. Alkyla ous examples of the SA crosslinking reagents that can be tion can also be achieved in the beginning by blocking the made using the methods and representative examples dis secondary OH groups with alkyl groups. closed herein. US 2015/001 7246 A1 Jan. 15, 2015

0644. To enable such strategies in FIGS. 2 and 3 the right removed by acid hydrolysis. THP protection can be achieved protection groups must be selected for the primary OH and by using any of the following catalysts: Zinc tetrafuloroborate secondary OH groups. The protecting groups (PG1 and PG2) (Ranu, B.C. etal. Tetrahedron Letters, 1999, 40, 1985-1988), need to be orthogonal in the deprotection conditions. Synthe pyridinium p-toleuensulphonate (Miyashita, M. et al J. Org. sizing a monofunctional and homobifunctional crosslinking Chem., 1977, 42, 3772-3774), bis(trimethylsilyl) sulfate reagent is often easier than synthesizing a heterobifunctional (Morizawa, Y. et al Synthesis 1981, 899-901), or amberlyst crosslinking reagent. For heterobifunctional crosslinkers, H-15 (Bongini, A. et al. Synthesis 1979, 618-620). PG1 has to be able to be selectively cleaved, as only one of the 0646. The general schemes and methods for introducing OH groups can be replaced with a crosslinking group at a crosslinking groups from the OH group are as follows. Mul time. tiple crosslinking groups can be introduced sequentially by 0645. Different protecting groups have been explored as releasing other OH groups one by one. Homobifunctional examples for obtaining a single OH-reactive SA molecule. 1) crosslinkers are synthesized when both PG1 groups are Tert-butyldimethylsilyl (TBDMS) ether protection of the pri deprotected.

Scheme 1: Converting an OH group to - ONH2. OPG2

PG11 sub-lpi

MsCI, Pyridine or B.

O OPG2 2. NHNH PG11 O N-- ONH 2 1. N-OH, Base . Y 121y 12 u- Aminooxy terminated I36 O

OPG2 /..no BocN-OHDBU PG11 pi I35

mary OH groups is easy to form and can one of the primary 0647. The single free OH group of the SA can first react OH groups can be selectively deprotected using dilute iodine with methanesulfonyl chloride and convert to mesylated OH solution. To selectively protect the primary OH, a more hin dered silylation reagent, t-butyl-diphenylsilyl chloride (TB (OMs). The alcohol mesylate group (OMes) is a very good DPSC1), was also explored. The reaction was much cleaner leaving good, and various nucleophiles (such as N3 and CN) than TBDMS protection. Unlike TBDMS, no indication was can easily attack and replace it. O-alkylation of tert-butyl seen that three protection groups were added. 2) Benzoate N-hydroxycarbamate (BocN-OH) with alcohol mesylate, fol ester is base sensitive and orthogonal to the TBDMS remov lowed by acidic N-deprotection, results in the corresponding ing conditions. 3) Benzyl ether is stable to acid and base, and hydrogenation is used to remove it. 4) THPether protection: aminooxy terminated SA (Albrecht, S. et al. Synthesis 2006, 3,4-dihydro-2H-pyran is known as a useful strategy for pro 10, 1635-1638.). Alternatively, the SA alcohol mesylate can tecting the primary and secondary OH groups to obtain tet react with N-hydroxyphthalimide-protected hydroxyamic rahydropyranyl ethers. THPether is stable in basic media and acid to create a compound that is deprotected under basic under oxidation and reduction conditions, but can easily be conditions (hydrazine). US 2015/001 7246 A1 Jan. 15, 2015 52

Scheme 2: Converting an OH group to an azide and amine.

1. NK 2. NHNH2

OPG2 OPG2 O N -e- PG11 N-'-pi 3 or Zn reductionO -O NH2 PG1 pi Azide terminated Amine terminated I37 I38 DPPA, P(Ph)3, DEAD, THF, 0° C.

OPG2 -N-- MsCl, Pyridine or Et3N

LiN, DMF sueyOPG2 PG11 pi I35

0648 AZide can be prepared in high yields via nucleo philic displacement of the corresponding mesylate or modi fied Mitsunobu conditions (triphenyl phosphine, diethyla Zodicarboxylate: DEAD; diphenylphosphoryl azide: DPPA) (Jackson, M. D. et al. J. Org. Chem. 2002, 67,2934-2941). The azide can be further reduced to an amine using different reducing reagents, such as Zn.

Scheme 3: Converting an OH group to CN, COOH, or N-hydroxysuccinimide (NHS) ester. OPG2 OPG2 O N-- OH --MsCI, Pyridine or EtN O N-- OMS PG11 pi PG11 pi

PPh3, DIAD, acetone NaCN cyanhydrin, THF

OPG2

PG11 Nuespi

NaOMe, Me OH, then aq. NaOH US 2015/001 7246 A1 Jan. 15, 2015

-continued O DCC OPG2 O OPG2

O Suely- N NHS-OH PG11 pi O1 PG11 Nu'ulopi Carboxy terminated I40

0649 OH can be directly converted to a CN group follow ing a procedure from the literature (Gollner, A.; Mulzer, J. Organic Letters, 2008, 10, 4701-4704) or via the mesylated Scheme 6: Converting an OH group to an alkyne. OPG2 alcohol intermediate. After hydrolysis of the CN group by MsCI, Pyridine or EtN acid or base (Bernardes, G. J. L. et al ChemBioChem 2011, -- 12, 1383-1386), a carboxylic acid can easily be obtained. The PG11 N-b-e'pi carboxylic acid can be activated further using dicyclohexyl carbodiimide (DCC) and N-hydroxysuccinimide to form an OPG2 NHS ester. CH-CCNa -e- PG11 --pi Scheme 4: Converting a carboxylic acid to a hydrazide. I35 OPG2 OPG2 1) 1-(methylsulfonyl) benzotriazole 1 Nu'ulo Her2) anhydrous PG11 N--~1 PG1 pi Hydrazine Alkyne terminated I44 Carboxy terminated I40 OPG2 O

PG11 Nues-pi N1 NH2 Scheme 7: Introducing a trifluoromethylphenyldiazirine via a H carboxylic acid group or amine group. Hydrazide I42 O OPG2 O > pH 8.0 O N Her 0650 A carboxylic acid group can be conveniently con PG11 pi O1 FC verted to a hydrazide in one pot via hydrazinolysis of the N intermediate N-acylbenzotriazole (Katritzky, A. R. O v ARKIVOC 2001 (ix) 19-23, issue in honor of academician I41 HN N Michael G.Voronkov). FC N f Scheme 5: Converting a CN group to an imidoester N OPG2 O OPG2 Anhydrous HCI/MeoH He PG11 subpi N PG11 Nullpi H I39 45 OPG2 OPG2 ON-- NH -->pH > 8.0 PG11 N-lespi 1 PG 11 pi O O NHCI I38 -- Amine terminated N O1 Imidoester FC I43 N O N N 0651 Imidoesters are highly specific reagents for amino groups in biopolymers. Imidoester can easily be prepared from a nitrile following procedures in the literature (McEl vain, S.M.; Schroeder, J.P.J. Amer: Chem. Sco. 1949, 71, 40; Davies, G. E.; Stark, G. R. Proceed of the National Academy of Sciences, 1970, 66, 651-656). US 2015/001 7246 A1 Jan. 15, 2015 54

-continued CF OPG2 7 --- N O I46

Scheme 8: Converting an OH group to a free SH and protected thiol. S S OPG2 MeO / N4. OMe % N/ PG11 N-th-olpi 1,2-dimethoxyethane, RT I3 OPG2 2,2-dithiodipyridine? OPG2 O N-- SH -e-MeOH O S PG11 pi -N1)-N-()s-V / I48

0652 OH can be converted to a free thiol in one pot using -continued 2.4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4- OPG2 disulfide (Lawesson’s reagent, LR) (Nishio, T.J. Chem. Soc. N-- 2 -e-DCCNHS-OH Perkin Trans 1993, 1113-1117). The free thiol can react fur PG11 pi O ther with 2,2'-dithio dipyridine in MeCH to give the disulfide I38 HO protected product (Jones, L. R. et al J. Am. Chem. Soc. 2006, 128,6526-6527). Other disulfide reagents, such as Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic) acid, or DTNB) can be introduced in a similar fashion. O O OPG2 N Scheme 9: Introducing a maleimide group via a carboxylic acid group or an amine group. N-- N PG11 pi r O O O Maleimide terminated I50 O 1n-N \ OPG2 O HN O O N -e- SA Macromolecules PG11 pi O1 > pH 8.0 O 0653. In one aspect, the present invention provides a col lective of novel SA macromolecules that can be used for the I41 modification of biomolecules, formulation of biologics, and drug delivery. In addition, Sophisticated three-dimensional architecture can be readily built into the system to make single pure SA macromolecules, such as linear, circular, O branched, and dendrimer SA macromolecules, depending on OPG2 O the requirement of the tethered molecules. These features enable modulation of the formulation and the physical prop PG11 sub-pi 1N-1 N \ erties of the final conjugates. Furthermore, this system may H be used to transport potent antitumor drugs to tumor tissue, O reducing undesirable side effects and frequency of dosing. 0654 SA macromolecules can be made from the SAback bone by (i) linking SAS linearly through the OH group of the backbone, (ii) cyclizing the SA unit at the termini, (iii) asym metrically branching the SA unit together, (iv) symmetrically branching the SA, or (V) repetitively branching SAS to build US 2015/001 7246 A1 Jan. 15, 2015

Sophisticated three-dimensional structures. In all cases, a group is introduced. If n is greater than 1 or the mono SA high MW (>1000 Da) crosslinking reagent or carrier mol building block of one X group is coupled several times, mul ecule can be made. tiple X groups can be introduced. The number of X and Y groups and the position of the X and Y groups can easily be Linear SA Macromolecule adjusted based on the mono SA building block and sequence of coupling. Methods of Synthesizing Linear SA Macromolecules 0660 Table 2 lists the molecular weight distribution of 0655 FIG. 6 illustrates an embodiment of a general examples of single MW SA macromolecules that can be approach for synthesizing SA building blocks containing synthesized based on the above methods. By using only four multiple SA units. The method consists of: (i) providing the types of Sugar alcohol monomer (where n is 2, 3, 4, or 5), after first SA unit with a free primary OH, (ii) substituting the nine iterations of the same sugar alcohol monomer, the MWs primary OH group with a good leaving group, (iii) providing of the obtained compounds range from 400 to 400,000 Da. the second SA unit with a free primary OH with the protective One can also fine tune the MW range by mixing and reacting group of the other primary OH (PG3) orthogonal to the pro with other monomers, dimers, trimers, tetramers, or higher tecting groups of the primary OH of the first SA unit (PG1), SA molecules. For example, when a tetramer of threitol (n=2) (iv) combining the first SA unit with the second SA unit under is mixed with a tetramer of mannitol (n=4), a MW of 1109 is conditions that permit the condensation of these two units, obtained. When a tetramer of xylitol (n-3) is mixed with a and (V) deprotecting one of the primary OH groups to provide tetramer of mannitol (n=4), a MW of 1230 is obtained. All of a di-SA building block (two SA units) that can be utilized to these molecules are made with chemically pure starting mate build a higher MW SA molecule. Higher orders of SA build rials bearing a single reactive site; only one coupling product ing blocks (higher number of SA units) can be built following can be obtained. This is the key to obtaining single pure SA repetitive steps (i) to (v) using r-SA building blocks, wherer molecules. represents the number of SA units in one molecule. 0656 Higher MW linear SA molecules can be built by TABLE 2 stepwise or convergent, or stepwise plus convergent, methods MW distribution of single MW SA macromolecules that can be depending on the heterogeneity of the secondary alcohol Synthesized by chemical methods. groups in the SA molecules. For example, tetra-SA building blocks containing different numbers and different kinds (dif OH OH ferent stereochemistry) of secondary OH groups on each SA X O Yl unit can be built by coupling four different mono-SA units pi iii. stepwise. Tetra-SA building blocks with different numbers and different kinds of secondary OH groups on each SA unit Item MW Iteration SA can also be built by coupling two units stepwise first, and then i (Da) in m w XI y1 i unit #) converging two of the di-SA building blocks together. Tetra 1 449 2 2 3 OH - ONH O 4 SA units with the same number and same kinds of secondary 2 570 3 3 3 OH - ONH O 4 OH groups on each SA unit can be built by coupling two 3 690 4 4 3 OH - ONH, O 4 di-SA building blocks (e.g., I20 plus I19). In most cases, the 4 810 S 5 3 OH - ONH O 4 5 866 2 2 7 OH - ONH 1 8 low MW SA building blocks are usually built stepwise and 6 1,106 3 3 7 OH - ONH 1 8 the high MWSAs areassembled by convergence. An embodi 7 1,346 4 4 7 OH - ONH, 1 8 ment of a general method for the convergent synthesis of 8 1586 5 5 7 OH - ONH 1 8 9 1,699 2 2 15 OH - ONH 2 16 tetra-SA building blocks is shown in FIG. 6. 10 2,179 3 3 15 OH - ONH 2 16 0657 FIG. 7 shows the calculated MW of linear SA mac 11 2,660 4 4 15 OH - ONH, 2 16 romolecules that can be synthesized by iterative coupling 12 3,140 5 5 15 OH - ONH 2 16 using tetra-D-mannitol as the building block. After the fifth 13 3,364 2 2 31 OH - ONH 3 32 14 4.325 3 3 31 OH - ONH 3 32 iteration, single and pure SA macromolecules over 20 KDa 15 5,286 4 4 31 OH - ONH, 3 32 can be readily synthesized from inexpensive starting materi 16 6,247 5 5 31 OH - ONH 3 32 als. 17 6,696 2 2 63 OH - ONH 4 64 0658. In one approach, SA macromolecules containing 18 8,617 3 3 63 OH - ONH 4 64 19 10,539 4 4 63 OH - ONH, 4 64 crosslinking groups X and Y at the terminican be synthesized 2O 12.461 5 5 63 OH - ONH 4 64 in a similar fashion as SA crosslinking reagents (method 21 13,358 2 2 127 OH - ONH 5 128 shown in FIG. 2 and FIG. 3). For example, tetra-SA mol 22 17,202 3 3 127 OH - ONH 5 128 ecules I22 and I23 can first react with a reagent that introduces 23 21,045 4 4 127 OH - ONH, 5 128 24 24,888 5 5 127 OH - ONH 5 128 functional group X. Then, after deprotecting the second pri 25 26,684. 2 2 255 OH - ONH 6 256 mary OH group, a second functional group, Y, can be intro 26 34,370 3 3 255 OH - ONH, 6 256 duced. Deprotection of the secondary OH groups produces 27 42,057 4 4 255 OH - ONH 6 256 tetra-SA crosslinking reagents. 28 49,744 5 5 255 OH - ONH 6 256 29 53,335 2 2 511 OH - ONH 7 512 0659 Functional groups X and Y may be incorporated into 30 68,708 3 3 511 OH - ONH, 7 512 either the primary OH groups at the termini or secondary OH 31 84,081 4 4 511 OH - ONH 7 512 groups at the side chains. FIG. 8 illustrates an embodiment of 32 99.454 5 5 511 OH - ONH 7 512 a general approach for incorporating X and Y groups into the 33 106,636 2 2 1023 OH - ONH 8 1024 34 137,383 3 3 1023 OH - ONH, 8 1024 SA macromolecules. A mono SA building block with the X or 35 168,129 4 4 1023 OH - ONH 8 1024 Y group already in place at the secondary OH groups is 36 198,876 5 5 1023 OH - ONH 8 1024 coupled to a SA macromolecule (I27). If n equals 1 and the 37 213,239 2 2 2047 OH - ONH 9 2048 mono SA building block is coupled only once, then one X US 2015/001 7246 A1 Jan. 15, 2015 56

TABLE 2-continued MW distribution of single MW SA macromolecules that can be synthesized by chemical methods.

OH OH X O Yl pi iii.

Item MW Iteration SA i (Da) in m w XI y1 i unit #) 38 274,732 3 3 2047 OH - ONH, 9 2048 39 336,225 4 4 2047 OH - ONH 9 2048 40 397,719 5 5 2047 OH - ONH 9 2048

Scheme 10:

CFSOOSOCF -e- Pyridine OBn OBn BZ = CH-CO Bn = CH3CH2CO

OTBDMS

OB OB

I2 MeOH OTBDMS -- i Bn OB OB

Higher MW SA Compound US 2015/001 7246 A1 Jan. 15, 2015 57

0661 Scheme 10 shows a general strategy for synthesiz (TLC, 25.75 EtOAc:hexanes; HPLC, 11.014 minutes using ing a higher MW sugar alcohol. One of the primary OH Method B), whereas the isomerized product has an R-value of groups of the mannitol is protected as a TBDMS ether and the 0.55 (HPLC: 11.182 min) (m/z expected for M+H=713.3, secondary OH group as benzyl ether. The remaining OH observed m/z713.6; m/z expected for M+Na=735.3, group is activated by reacting with trifluoromethanesulfonic acid anhydride in pyridine. Another mild leaving strategy can observed m/z =735.6; m/z expected for 2M--Na-1447.6, also be used, such as O-mesylation. This pre-activated man observed m/z =1448.2). In contrast, the benzyl ether protec nitol carrying a good leaving group can couple to another tion was very stable under the dimerization reaction condi mannitol carrying a single free OH group in the presence of a tions during a separate run using threitol as an example and base, such as NaH, LiHMDS, or potassium tert-butoxide. The was easily removed during hydrogenation. The above strat initial strategy of using benzoate ester as the starting material egy uses benzyl ether protection instead of benzoate ester (compound I3-a) for the dimerization was unsuccessful under protection. The only drawback of the above strategy is that the similar conditions. Apparently, the benzoate ester of I3-a yield for selective iodine deprotection of the TBDMS is not underwent intramolecular isomerization during the reaction very high (-50%) based on previous experiments, which will with little or no dimer formation. I3-a has an R-value of 0.43 affect the overall efficiency of multiple synthesis.

Scheme 11: OBn OBn OH HO OBn OBn S

NaHFTHF8. C ul

OBn OBn ODMTC HO OBn OBn CFSOOSOCF3 v-2 2V-13 OBn OBn A.O OH O-Si-CF OB OB TBDMSO Pyridine TBDMSO Y NaH, THF OBn OBn OBn OBn BZ = C6H5-CO Bn = CH5CH2CO

OBn OBn OBn OBn OBn OBn OBn OBn O I2;MeOH O TBDMSO ODMTC -- OH ODMTC OBn OBn OBn OBn OBn OBn OBn OBn 15 Atom spacer: Di-mannitol into

Higher MW SA Compound US 2015/001 7246 A1 Jan. 15, 2015

0662 Scheme 11 outlines another strategy in which a third the core is an SA unit and other multiple SA (mSA) building protecting group. Such as DMTC (dimethylthiocarbamate), is blocks are attached to all of the OH groups at the core SA unit. used. DMTC is stable with a wide range of reagents and In some embodiments hyperbranched SA macromolecules conditions, including metal hydrides, acid, base, and heat. It have a dendrimer-like configuration wherein the SA macro is easily removed by NaIO or HO and the other common molecules continue to grow through the branches of the tree alcohol protecting groups (Barma, D. K. et al. Organic Let like SA macromolecules. In another embodiment, hyper ters, 2003, 5,4755-4757). This property will greatly increase branched SA macromolecules have a pyramid-like the efficiency of the synthesis. configuration wherein the core is a glycerol unit. Branching 0663 Linear SA macromolecules can also be synthesized can also occur to the side chain of the cyclic SA macromol using the Solid phase approach by coupling mono SA units or ecule as shown in the few configurations in FIG. 10. FIG. 11 r-SA units (multiple SA units) one by one. For example, an shows examples of Some actual branched molecules (e.g., SA unit containing an Fnoc-protected amine at one terminus symmetrical, asymmetrical, linear, and cyclic) based on and a carboxylic acid at another terminus may be synthesized D-mannitol. first, then the linear SA macromolecule can easily be synthe 0669 Hyperbranched molecules, such as dendrimers, sized following the Fmoc Solid phase strategy similar to pep have been found to have the characteristics of an ideal drug tide synthesis. In another example, an SA unit containing a delivery vehicle and have been widely investigated as poten DMT-protected amine at one terminus and a phosphoramidite tial carriers of drugs, genes, and vaccines (Patri, A. K. et al. at another terminus can be synthesized first, then the linear Curr. Opin. Chem. Biol. 2002, 6, 466-471.; Qivand, L.Y. and SA macromolecule can be assembled following the standard Bae, Y. H. Pharm. Res. 2006, 23, 1-30; Al-Jamal. K.T. et al. oligonucleotide synthesis strategy. FIG. 9 provides two J. Pharm. Sci. 2005, 94, 102-113). The synthesis of a den examples of Such linear SA macromolecules synthesized by drimer was first reported in 1985 (Tomolia, D. A. et al. Polym. the Solid phase strategy. J. (Tokyo), 1985, 17, 117-132; Newkome, G. R. et al. J. Org. Chem. 1985, 50, 2003-2004). The details of dendrimer syn Cyclic, Branched, Hyperbranched Macro SA Molecules thesis have been reviewed extensively (Bai, S. et al. Crit. Rev. 0664. In another aspect, the invention generally relates to Ther. Drug Carrier Syst. 2006, 23, 437-495). a precursor compound that is a macrocyclic SA molecule comprising three or more monomeric SA units, B' (formula Methods of Synthesizing Branched SA Macromolecules III), with each monomericSA unit connected linearly through 0670 Branched SA macromolecules can be synthesized the X and Y portion of the SA unit one by one and then by incorporating linear SA macromolecules at the side chain cyclized through the reaction of the X group of the first SA of the monomeric SA unit (secondary OH groups). FIG. 12 unit to the Y group of the last SA unit. In certain embodiments illustrates embodiments of a general approach for synthesiz the SA units of the macrocyclic SA molecule are cyclized ing a branched SA macromolecule. The method consists of through the reaction of the X group of the first SA unit to the (i) providing the first SA unit with a free secondary OH group Z group of the last SA unit. In certain embodiments the SA while the primary OH groups are protected with PG1, (ii) units of the macrocyclic SA molecule are connected through Substituting the secondary OH groups with a good leaving the Z group of any SA in any order but in a cyclized form. group, (iii) providing the second linear SA macromolecule 0665. The precursor compound may be a branched SA building block with a free primary OH while the secondary macromolecule comprising three or more monomeric Sugar OH groups are protected with PG2 and the other primary OH alcohol units, B', with each monomeric Sugar alcohol unit is protected with PG3, which is orthogonal to the protecting bound to one or another monomeric Sugar alcohol unit group (PG1) of the primary OH of the first SA unit, (iv) through a linking group, W, wherein at least one of the linking combining the first SA unit with the second linear SA mac groups is formed by a reaction between the X, Y, or Z portion romolecule under conditions that permit the condensation of of one monomeric unit with the Z of another monomeric unit. these two units, and (V) deprotecting one or two of the primary 0666. The precursor compound may be a three-dimen OH groups to provide sites for further branching. The mac sional hyperbranched SA macromolecule comprising six or romolecule is usually non-symmetrical (e.g., P19). When a more monomeric Sugar alcohol units, B', in which the core is glycol is used as the core SA unit, the macromolecule is an SA unit and several SA units branch out from the core symmetric (e.g., P20). structure. The hyperbranched SA macromolecule is largely a 0671 Small MW SA homobifunctional and heterobifunc monodispersed, three-dimensional molecule with a well-de tional crosslinking reagents may be used to crosslink differ fined MW. The characteristic hyperbranched SA macromol ent organic molecules or biomolecules together. Similar con ecule is a multiple branching molecule in three dimensions. jugates created with other crosslinking reagents have found When the size of the hyperbranched SA macromolecule many uses as research tools in biomedical research, as diag approaches nano or micro scales, or microsphere may be nostic reagents, and as new pharmaceutical drugs. High MW obtained. SA crosslinking reagents can be used to modify therapeutic 0667. In certain embodiments the OH groups in an SA agents and as carriers for drug delivery. For example, high macromolecule (cyclic, branched, and hyperbranched) are MWSA macromolecules can be used to conjugate proteins of unmodified and can be used directly to link molecules of pharmaceutical interest that have a MW of less than 30 KDa, interest through the OH groups. In some embodiments one or Such as enzymes, cytokines, hormones, and monoclonal anti more of the OH groups may be replaced with X, Y, or Z body fragments. In addition, SA macromolecules may be crosslinking groups. Molecules of interest can be conjugated used to conjugate peptides, nucleic acids and their analogs, by reacting with X, Y, or Z crosslinking groups. and siRNAs. The benefit of conjugating such high MW SA 0668 FIG. 10 lists a few configurations of hyperbranched macromolecules to these molecules include (i) an increased SA macromolecules. In some embodiments hyperbranched hydrodynamic Volume of the conjugate molecules, reducing SA macromolecules have a tree-like configuration wherein their kidney excretion and prolonging the in vivo half-life of US 2015/001 7246 A1 Jan. 15, 2015 59 proteins to stabilize the protein, (ii) protecting amino acids of bridge to which the SA molecule is covalently attached peptides from degradation, (iii) masking the critical sites (Shaunak S. et al. Nat. Chem. Bio. 2006,312-313; Balan S. et sensitive to enzymatic degradation, (iv) reducing aggrega al., Bioconjugate Chem. 2007, 18, 61-76.; Brocchini, S. et al. tion, and (V) increasing bioavailability. Nat. Protocols. 2006, 1, 2241-2252). 0672. In addition, SA macromolecules can be metabolized 0678. In some embodiments various enzymatic methods in Vivo, which may generate a new mechanism for drug can be used to label molecules of therapeutic interest in a release. site-specific manner using SA macromolecules. The enzymes 0673. Furthermore, SA macromolecules may be used to recognize and specifically modify only a select single or few load drugs or Small molecules, not only at the termini, but amino acid residues, such as glycosyl-transferases and trans throughout the SA macromolecule. For example, molecules glutaminases. containing acid groups can be coupled to the OH group of the 0679. In some embodiments X or Y is a sialic acid group backbone, increasing the amount of Small molecules deliv linked either directly to the SA macromolecules or through ered to the tissue. other spacers. Site-specific labeling can be achieved through enzymatic GalNAc glycosylation at serine and threonine resi Site-Specific Labeling Through SA Crosslinking Reagents dues in the protein, followed by the enzymatic transfer of and SA Macromolecules SA-linked sialic acid to the previously introduced GalNAc residues. GlycoPEGylation has been applied successfully to 0674 Site-specific labeling may beachieved through vari the modification of granulocyte colony Stimulating factor ous strategies: (i) terminal amino acid in the protein of inter (G-CSF) (Defrees, S. US Patent Publication No. est, (ii) insertion of a non-paired cysteine as a labeling site, 20070254836A1), granulocyte macrophage colony stimulat (iii) structure-based approach by predicting the most reactive ing factor (GM-CSF) (DeFrees, S. et al. Glycobiology, 2006, group in the Surface. Such as amine, phenolic hydroxyl, and 16, 833-843), interferon alpha-2b (IFNC-2b) (DeFrees, S. et thiol, (iv) oxidation or enzymatic reaction of a glycosylated al. Glycobiology, 2006, 16,833-843), follicle stimulatinghor moiety on the protein followed by tethering, (v) insertion of mone (DeFrees, S. et al. US patent application unnatural amino acids into the protein sequence to enable 2008.0015142), erythropoietin (DeFrees, S. et al. US Patent labeling at that specific site, and (vii) cysteine mutagenesis to Publication No. 20060287224A1), and factor VII (Klausen N introduce single reactive CyS groups. Ketal. US Patent Publication No. 20080039373A1). 0675 SA crosslinking reagents may be used for the site 0680. In some embodiments X orY is an amine functional specific labeling of molecules of interest. For example, an SA group linked either directly to the SA macromolecules or molecule can label only the N-termini of proteins, antibodies, through another spacer/linker. The conjugation reaction of a and enzymes through reductive amination. In some embodi single amine SA macromolecule is achieved through trans ments, the X orY groups are aldehyde functional groups. For glutaminase, where the amino group of the SA macromol example, this strategy was used for site-specific PEGylation ecule serves as a donorand glutamine in the protein or peptide (Lee, D. et al. J. Interferon Cytokine Res. 2008, 28, 101-112). serves as an acceptor. This process has been used to conjugate An example of a conjugate made by this chemistry is Neu PEG to granulocyte-colony stimulating factor (BK0026) lasta R (PEG-filgrastim) (Kinstler O B. Pharm. Res. 1996, 13, (Tonon G. 2008, WO 2008/017603), interleukin-2 (Sato H. 996-1002.). Adv. Drug. Deliv Rev. 2002, 54, 487-504), growth hormone 0676 In some embodiments, thiol-specific SA crosslink (Zundel M. 2006, WO2006/084888), and erythropoietin ing reagents or SA macromolecules are used for site-specific (Pool CT, 2004, WO 2004/108667). labeling of Cys in the protein, with which single reactive Cys can be obtained by CyS mutagenesis. SA crosslinking SA Crosslinked Conjugates reagents or SA macromolecules can undergo thiol eXchange reactions or alkylation reactions through ether Substitution 0681. The SA macromolecules herein may be used to con (such as iodoacetiamide derivatives) or an addition reaction jugate and link one molecule to another to produce new (such as a vinyl sulfone, vinyl pyridine, or maleimide end homogeneous or heterogeneous chemical entities. Here, any group). of the X, Y, Z, or ketone groups can react with one or a few crosslinking groups of the other molecule of interest to form 0677. In some embodiments X or Y is a conjugation moi a conjugate. ety with the formula 0682. In one aspect, the invention generally relates to a conjugate having a chemical structure selected from the R10 group consisting of: O V M-(L-B). Formula (I), and S R111 V B-(L-M), Formula (II) O 0683 wherein 0684 each M is independently selected from the group wherein R'' is an optionally substituted aryl and R' is an consisting of a protein, an enzyme, an antibody, an antibody electron withdrawing group (e.g., carbonyl), an O.f3-unsatur fragment, a polypeptide, an oligonucleotide, an oligonucle ated double bond, oran C.B Sulfonyl group prone to elimina otide analog, a polysaccharide; tion as Sulfonic acid. This compound can be used to label any 0685 each B is a single MW modified sugar alcohol poly protein or antibody with a disulfide bond in a site-specific mer, comprising: manner through two steps: (i) mild disulfide reduction to 0686 from 2 to about 2000 sugar alcohol monomer(s): release the free thiols and (ii) conjugation of both thiols by 0687 wherein each monomer has from 3 to about 14 sequential interactive bis-alkylation to yield a three-carbon —OR' groups; US 2015/001 7246 A1 Jan. 15, 2015 60

0688 wherein R' is independently selected from 0702. In another aspect, the invention is generally relates the group consisting of hydrogen, C-C alkyl, ben to a conjugate having chemical structural Formula (III): Zoyl, acetyl, benzyl, C-C alkylsilyl cyclic ortho ester, actinide, acetate, tetrahydropyranyl, tetrahy (M-L)-B (III) drofuranyl, and tetrahydrothiofurany; 0703 wherein 0689 each L is independently selected from the group 0704 each M is independently selected from the group consisting of: consisting of a metabolite, a fluorescent compound, a chemi (0690 a Rand, a structure of V. R V , wherein: luminescent compound, a mass tag, a chromophore, biotin, a (0691 V and V are independently selected from the toxin, a drug, a chemotherapeutic agent, a cytotoxic agent, an group consisting of: immunosuppressive agent, a diagnostic agent, a radioligand. 0692 Diels-Alder adduct, a 1,3-dipolar adduct, and a small molecule, —C(=G)-G-, -G-C(=G')-, -G-, -G-C(=G')-G-, 0705 each B is a single MW modified sugar alcohol poly mer, comprising: 0706 from 2 to about 2000 sugar alcohol monomer(s): 0707 wherein each monomer has from 3 to about 14 —OR' groups; (0708 wherein R' is independently selected from the group consisting of hydrogen, C-C alkyl, ben Zoyl, acetyl, benzyl, C-C alkylsilyl cyclic ortho ester, actinide, acetate, tetrahydropyranyl, tetrahy drofuranyl, and tetrahydrothiofurany; 0709 each L is independently selected from the group consisting of: (0710 a R and, a structure of V. R. Vs wherein: 0711 V and V are independently selected from the group consisting of: 0712 Diels-Alder adduct, a 1,3-dipolar adduct, —C(=G)-G-, -G-C(=G)-, -G-, -G-C(=G)-G-, S-S , —S-(CH2). S(O) , —S(O) (CH), S , S(O), N(R)—, N(R) S(O) , —C(O) NH NH-CH , —C(O) NH N=CH-, -C H=N. NH C(O) , —CH NH NH CO) , N(R) S(O), N(R) , 3-7. —C(O) NH-CH(CHSH) , - N=CH-, NH-CH , NH C(O)-CH C(O) NH CH-N-G-CH NH-G-G-NH CH-, -G-N=CH-, -C(=NH"). NH-, NH CO-NH) - O P(=O)(O) NH , -NH P(=O)(O)-O-, -CH-CH(NH)– CH2—S—, —S CH-CH(NH)—CH2—, - O P(=O)(O)-O-, - O P(=O)(S)-O , a - O P(=S)(S)-O , , the 0693 wherein: (0694 each G' is independently selected from NR, 3-7. O, and S; (0695 each G’ is independently O or S: (0696 each G is independently selected from S, O, NR, and SO: (0697 each G' is independently O or NR; (0698 each R is independently selected from a bond, C-C alkyl, -(CH2CH2O). , —(CH2CH2O). a 10—CH2—, optionally Substituted alicyclyl, heteroali cyclyl, aryl, a peptide, and a peptidomimetic oligomer, (0699 each Risindependently selected from hydrogen, C-Cs alkyl, —(OCH2CH2), optionally substituted alicyclyl, and optionally substituted heteroalicyclyl; (0700 each R is independently C-Cs alkyl: , the 0701 u is an integer from 1 to about 20; and, US 2015/001 7246 A1 Jan. 15, 2015

0713 wherein: 0714 each G' is independently selected from NR, O, and S; 0715 each G' is independently O or S: 0716) each G is independently selected from S, O, NR, and SO: 0717 each G' is independently O or NR; 0718 each R is independently selected from a bond, C-C alkyl, -(CH2CH2O)o , —(CH2CH2O). 10—CH2—, optionally Substituted alicyclyl, heteroali 3-7. cyclyl, aryl, a peptide, and a peptidomimetic oligomer, (0719 each Risindependently selected from hydrogen, C-C alkyl, -(OCH2CH), optionally substituted alicyclyl, and optionally substituted heteroalicyclyl; 10720 each R" is independently C-Cs alkyl: 0721. In another aspect, the invention is generally relates to a conjugate having a chemical structure selected from the group consisting of a (M1)-L-(B-(L-M2)), (IV) and (M-L)-(B-(L-M2)), (V) and M-(L-B-(L-M-2)), (VI) , the 0722 wherein 0723 each M is independently selected from the group 0733 wherein: consisting of a protein, an enzyme, an antibody, an antibody fragment, a polypeptide, avidin, Streptavidin, an oligonucle 0734 each G' is independently selected from NH, O, otide, an oligonucleotide analog, a polysaccharide; and S; 0724 each M is independently selected from the group 0735 each G’ is independently O or S; consisting of a metabolite, a fluorescent compound, a chemi (0736 each G is independently selected from S, O, luminescent compound, a mass tag, a chromophore, biotin, a NR, and SO: toxin, a drug, a chemotherapeutic agent, a cytotoxic agent, an (0737 each G' is independently O or NR; immunosuppressive agent, a diagnostic agent, a radioligand. (0738 each R is independently selected from a bond, a small molecule, and another biologically active molecule: C-C alkyl, -(CH2CH2O). , —(CH2CH2O). 0725 each B is a single MW modified sugar alcohol poly 10—CH2—, optionally Substituted alicyclyl, heteroali mer, comprising: cyclyl, aryl, a peptide, and a peptidomimetic oligomer; 0726 from 2 to about 2000 sugar alcohol monomer(s): 0739 each R is independently selected from hydrogen, 0727 wherein each monomer has from 3 to about 14 C-Cs alkyl, —(OCH2CH2), optionally Substituted —OR' groups; alicyclyl, and optionally substituted heteroalicyclyl; 0728 wherein R' is independently selected from 0740 each R is independently C-C alkyl: the group consisting of hydrogen, C-C alkyl, ben Zoyl, acetyl, benzyl, C-C alkylsilyl cyclic ortho 0741 u is an integer from 1 to about 100: ester, actinide, acetate, tetrahydropyranyl, tetrahy 0742 q is an integer from 1 to about 100; and, drofuranyl, and tetrahydrothiofurany; 0743 k is 0 or an integer from 1 to about 20. 0729 each L is independently selected from the group 0744. In some embodiments, q is an integer from 1 to consisting of: about 10 and B comprises from 3 to about 2000 sugar alcohol (0730 a Rand, a structure of V. R V , wherein: monomers. In some embodiments, u is an integer from 1 to (0731 V and V are independently selected from the about 10 and B comprises: from 3 to about 2000 sugar alcohol group consisting of: OOCS. 0732 Diels-Alder adduct, a 1,3-dipolar adduct, 0745. In some embodiments, M is selected from the —C(=G)-G-, -G-C(=G)-, -G-, -G-C(=G)-G-, group consisting of an antibody and an antibody fragment; and M is a chemotherapeutic drug, q is 1; u is an integer from 1 to about 20; and q is an integer from 1 to about 10. 0746. In another aspect, the invention is relates to a con jugatehaving chemical selected from the group consisting of: US 2015/001 7246 A1 Jan. 15, 2015 62

0747 wherein -continued 0748 S comprises a solid support; O O 0749 each M is independently selected from the group consisting of a protein, an enzyme, an antibody, an anti body fragment, a polypeptide, avidin, Streptavidin, an N N oligonucleotide, an oligonucleotide analog, a polysac ?y h charide; O , and O , 0750 each M is independently selected from the group consisting of a metabolite, a fluorescent compound, a chemiluminescent compound, a mass tag, a chro 0759 wherein: mophore, biotin, a toxin, a drug, a chemotherapeutic 0760 each G' is independently selected from NH, agent, a cytotoxic agent, an immunosuppressive agent, a O, and S; diagnostic agent, a radioligand, a small molecule, and 0761 each G’ is independently O or S; another biologically active molecule; 0762 each G is independently selected from S.O. 0751 each B is a modified sugar alcohol polymer, com NR, and SO: prising: 0763 each G' is independently O or NR; 0752 from 2 to about 2000 sugar alcohol monomer 10764 each R is independently selected from a bond, (s): C-C alkyl, -(CH2CH2O). , —(CH2CH2O). 0753 wherein each sugar alcohol monomer has 10—CH2—, optionally Substituted alicyclyl, het from 3 to about 14 —OR' groups: eroalicyclyl, aryl, a peptide, and a peptidomimetic (0754 wherein R' is independently selected oligomer, from the group consisting of hydrogen, C-Cs 10765 each R is independently selected from hydro alkyl, benzoyl, acetyl, benzyl, C-C alkylsilyl, gen, C-C alkyl, —(OCH2CH2), optionally Sub cyclic ortho ester, actinide, acetate, tetrahydro stituted alicyclyl, and optionally substituted het pyranyl, tetrahydrofuranyl, and tetrahydrothio eroalicyclyl; furany; 10766) each R" is independently C-Cs alkyl: 0755 wherein each L is independently selected from 0767 u is an integer from 1 to about 500; and, the group consisting of a R and —V-R V , 0768 k is 0 or an integer from 1 to about 20. (0756 wherein: 0769 Solid support includes, for example, agarose beads, 0757 V and V are independently selected from dextran, silica gel-based polymer, polystyrene, PLGA, the group consisting of PMMA, and colloidal gold. S may be a microparticle. The 0758 Diels-Alder adduct, a 1,3-dipolar adduct, chemical composition of the particles may be polymers or —C(=G)-G-, -G-C(=G')--G-, -G-C(=G)- copolymers, inorganic constructs, metals and semiconduc G'-, -S-S-, -S-(CH2), S(O) , tors, Superparamagnetic composites, biodegradable con S(O) (CH). S. , S(O), N(R) —, structs, or synthetic dendrimers and dendrons. S may be N(R) S(O) , C(O) NH NH buckyballs, fullerenes, or carbon nanotubes. S may also be a CH2—, C(O) NH N=CH-, quantum dot, dye-coded particle, or magnetic-coded particle. CH=N. NH C(O) , CH NH 0770. In some embodiments, each B is a single MW modi NH-C(O) , N(R) S(O), N(R) , fied sugar alcohol polymer, comprising from 2 to about 2000 —C(O) NH-CH(CHSH) , N=CH-, Sugar alcohol monomer(s) and each Sugar alcohol monomer NH-CH - NH C(O)-CH C(O) is bound to one or more Sugar alcohol monomers through a NH-, -CH=N-G-, -CH NH-G-, -G- linking group W formed by a reaction between the X, Y or Z NH-CH , -G-N=CH-, -C(=NH)– portion of one monomeric unit with the X, Y or Z of another NH , NH CO-NH) - O P(=O) monomeric unit; (O) NH , NH P(=O)(O) O , 0771 wherein —CH2—CH(NH)—CH2—S— —S CH (0772 for each B", independently, has the chemical struc CH(NH) CH-, - O P(=O)(O) O , tural Formula XI: - O P(=O)(S)-O-, - O P(=S)(S)- O—,

(XI)

3-7. s 0773 each of n and p is independently selected from 0 and an integer selected from 1 to about 12, and n+p is between 1 and 12; 0774 r is 0 or 1; 0775 each bond represented by ... is a single or a double bond; US 2015/001 7246 A1 Jan. 15, 2015 63

(0776 Q is selected from —S CH-CH(NH) CH-, - O P(=O)(O)— —N—O-L-M =N-O-L-S, O - O P(=O)(S)-O-, and - O P(=S) O-L-M, and NH-O-L-M, (S)-O , (0777 each of X, Y and Z., when bound to M, M, or S, is a linker V. (0778 each of X, Y and Z., when not bound to S or M, M, is a functional group that independently selected from -OH,-J.-RJ, —C(=O)-J, —C(=O)—CH-J, NH C(=O) CH-J, OR, OR, OR, O Mesyl, —O-Tosyl, - NH C(=O)—CH2—O-Mesyl, —NH CO =O)—CH2—O-Tosyl, —SH, S S-tBu 3-7. tyl, - SR7, -SR,-S-S-R, -S(=O).-J., NH, NHR, N(R)R, NHR'. NH-Fmoc, NH Boc, -C(=O)H, -C(=O) R, —C(=O)CH, N–C–S, N–C–O, C=C R, N-N- —N, O NH, O. NH-Fmoc. - O NH-Boc, —O N-(Boc) —O N(-phthalimidyl), NH NH, C(=O) NH NH, -NH CO-O) NH NH, -NH C(=S) NH NH, -toluenesulfonyl a hydrazide, R NH C(-NH) NH, a benzophenone, an aryl diazonium, a diazoalkane, a dia Zoacetyl, an anthraquinone, a diazirine, an optionally substituted trifluoromethylphenyldiazirine, a diene, a dienophil, a 1,3-dipole, a dipolarophile, an alkene, a , the ketene, an olefin, an alkene with allylic hydrogen, a dicarbonyl group, an epoxide, an oxirane, an organosi 0787 wherein: lane, a phosphonium group, an ester, an anhydride, a 0788 each G' is independently selected from NR, carbonate group, a glyoxal, —C(=NH)-O-R, a O, and S; hydroxymethyl phosphine derivative, an ethyl vinyl, a 0789 each G' is independently O or S: maleimide, a vinylsulfone, anallyl Sulfone, a thioester, a 0790 each G is independently selected from S, O, cisplatin derivative, an aziridine, an acryloyl group; NR, and SO; (0779 each Risindependently selected from hydrogen, 0791) each G' is independently O or NR; C-Cs alkyl, alicyclyl, heteroalicyclyl, benzyl or aryl, 0792 each R is independently selected from a bond, wherein any ring in R is optionally substituted; C-C alkyl, -(CH2CH2O)o , —(CH2CH2O). lo—CH2—, optionally substituted alicyclyl, het 10780 each R is independently selected from benzoyl, eroalicyclyl, aryl, a peptide, and a peptidomimetic acetyl, benzyl, C-C alkylsilyl, tetrahydropyranyl, tet oligomer, rahydrofuranyl, and tetrahydrothiofuranyl: 0793 each R is independently selected from hydro 10781) each R" is independently selected from trityl, gen, C-C alkyl, —(OCH2CH2), optionally Sub MMT, and DMT: stituted alicyclyl, and optionally substituted het 0782 each R is independently selected from 2-pyridyl, eroalicyclyl; 4-pyridyl, 5-nitro-2-pyridyl, 5-nitro-4-pyridyl, 2-nitro 0794 each R is independently C-C alkyl: phenyl, 4-nitrophenyl, 3-carboxy-4-nitrophenyl, and 0795 each R is a bond or -CH : 2,4-dinitrophenyl; 0796 and at least in one of the B' unit each -L-Z 0783 each J is independently selected from Cl, Brand portion is —OR'; I 0797 wherein each R" is independently selected from 0784 each of L', L, and L is independently a R or the group consisting of hydrogen, C-C alkyl, benzoyl, -R V R *, wherein: acetyl, benzyl, C-C alkyl silyl, cyclic ortho ester, 0785 66: represents a portion of L', L, and Li actinide, acetate, tetrahydropyranyl, tetrahydrofuranyl, bound to X, Y. S. Mor M, or a Z, respectively; and tetrahydrothiofurany. 0786 each W and V are independently selected from the 0798 In some preferred embodiments B has the chemical group consisting of Diels-Alder adduct, a 1,3-dipolar B has the chemical structural Formula XII: adduct, —C(=G)-G-, -G-C(=G)-, -G-, -G-C (=G)-G'-, - S S , —S (CH), S(O), s (XII) S(O) (CH), S , S(O), N(R) s N(R) S(O) , C(O) NH NH CH, s C(O) NH N=CH-, -CH=N. NH C(O) , CH-NH-NH C(O) , N(R) S(O), N (R) -, -C(O) NH-CH(CHSH) , N=CH-, NH-CH , -NH C(O)-CH C(O) NH-, CH=N-G-, -CH NH-G-, -G -NH-CH , -G-N=CH C(-NH) NH , NH C (—NH) - O P(=O)(O) NH NH P (=O)(O)—O , —CH2—CH(NH)—CH2—S , US 2015/001 7246 A1 Jan. 15, 2015 64

0799 wherein represents a bond to L'; and x c.2 represents a bond to L. represents a bond to M, M, or S. 0809. In some embodiments, when each of q and k is 1, (0800 each of L', when bound to M, Mor S, is a linker and u is larger than 1, the conjugate has the formula M-L- V (B-L-M), or. M-(L-B-L-M.). In some preferred embodi 0801 when k is 0, ments B is a sugar alcohol modifier with a MW.<1000, for example, an antibody linked to several cytotoxic drugs through Sugar alcohol moiety B, or a fluorescence-labeled protein or enzyme through Sugar alcohol crosslinking 2 reagent. 0810. In some embodiments, when k is 0 and q is 1, the conjugate has the formula M-L-B. M is simply labeled with different numbers of modifier B, which can further react with other molecules of interest to form a conjugate, M-(L- B-L-M). In certain preferred embodiments B is a Sugar represents Y. when k is an integer selected from 1 to about 20, alcohol modifier with a MW-1000. 0811. In some embodiments, when k is 0, u is 1, and q is larger than 1, the conjugate has the formula (M)-L-B, B-(L- 2 M), or (M-L)-B. In some preferred embodiments B is an SA macromolecule with MW-1000 Da (e.g., a drug carrier). Polymeric microspheres, polymer micelles, soluble poly c. mers, and PEGylation have been shown to be effective in enhancing drug stability and target specificity and decreasing toxicity. SA macromolecules can be used to pharmaceutically represents a bond to M, M, or S. load useful groups or entities, then deliver the drug in vivo. 0802. In another preferred embodiment B has the chemi 0812. In some embodiments, when k is 0, u is 1, and q is 1, cal structural Formula XIII: the conjugate has the formula M-B or M-B. In a preferred embodiment B is an SA macromolecule with MW >10000 * I-L4-(B' W), B-Li-2 (XIII) Da, wherein B can carry various large MW biomolecules, 0803 wherein: Such as proteins, siRNAS, oligos, peptides, and polypeptides. 0813. In certain embodiments M is a biomolecule, a (0804 “*” represents a portion of the L'bound to S, M. metabolite, a fluorescent compound, biotin, a toxin, a drug, a or M: chemotherapeutic agent, a diagnostic agent, or other biologi (0805 each of L', when bound to M, Mor S, is a linker cally active molecule. V 0814. In certain embodiments Mor M may include one 0806 s is 0 or an integer independently selected from 1 or more radioactive isotopes or isotopic elements. to about 500; 0815. In certain embodiments M is a metabolite. Metabo lites are the intermediates and products of metabolism. (0807 when k is 0, L*-* represents Y; and when k is an Examples of metabolites include alkaloids, glycosides, lip integer selected from 1 to about 20, “*” represents a ids, flavonoids, nonribosomal peptides, phenazines, phenols, portion of the L' bound to M, M, or Sand. polyketides, terpenes, and tetrapyrroles. Metabolites may (0808 each B", independently, has the chemical structural also be fragments of drugs or drugs modified by living organ Formula XIV: isms through specialized enzymatic systems. 0816. In certain embodiments the conjugate is an anti body-drug conjugate. M is an antibody orantibody fragment

(XIV) and M is a chemotherapeutic drug. From 1 to 8 drug mol ecules may be connected to a single antibody. 0817. In some preferred embodiments M and M is a therapeutic agent, Such as a drug, toxin, cytokine, hormone, hormone antagonist, enzyme, enzyme inhibitor, inhibitory oligonucleotide (e.g., RNAi, siRNA), immunomodulator (e.g., cytokine, lymphokine, chemokine, growth factor, or tumor necrosis factor), radionuclide, anti-angiogenic agent, pro-apoptotic agent, antibody, radiolabeled antibody, or pho toactive therapeutic agent. 0818. In certain embodiments the therapeutic agent is a chemotherapeutic drug. Examples of chemotherapeutic drugs include adrenocortical Suppressants, antimetabolites, alkylating agents, alkyl Sulfonates, antibiotics, antimitotics, US 2015/001 7246 A1 Jan. 15, 2015 anthracyclines, anti-angiogenic agents, camptothecins, Staphylococcal enterotoxin-A, pokeweed antiviral protein, COX-2 inhibitors, CPT-11, doxorubicin, doxorubicin ana gelonin, diphtheria toxin, Pseudomonas exotoxin, and logs, enzyme inhibitors, endostatin, epipodophyllotoxins, Pseudomonas endotoxin. ethylenimine derivatives, folic acid analogs, gemcitabine, 0824. In some preferred embodiments the therapeutic HDAC inhibitors, heat shock protein (HSP) inhibitors, hor agent is an immunomodulator selected from the group con mone antagonists, methotrexate, methyl hydrazine deriva sisting of cytokines, stem cell growth factors, lymphotoxins, tives, mTOR inhibitors, nitrosoureas, nitrogen mustards, hematopoietic factors, colony stimulating factor (CSF), inter pyrimidine analogs, purine analogs, platinum coordination ferons (IFNs), erythropoietin, thrombopoietin, and a combi complexes, Substituted ureas, SN-38, taxols, triaZenes, tax nation thereof. anes, tyrosine kinase inhibitors, proteosome inhibitors, pro 0825 M may be any antibody or fragment that is capable apoptotic agents, and Vinca alkaloids. Suitable chemothera of binding specifically to a target antigen associated with a peutic agents are described in the literature (Remington's disease state or condition. Antibody sequences or antibody Pharmaceutical Sciences, 19" Ed. Mack Publishing Co. secreting hybridomas against almost any disease-associated 1995; Goodman and Gilman's the Pharmacological Basis of antigen may be obtained from ATCC (AmericanType Culture Therapeutics, 7" Ed. McMillan Publishing Co. 1985). Collection), NCBI, and USPTO databases. Useful polyclonal 0819. In certain embodiments the therapeutic agent is a antibodies are heterogeneous populations of antibody mol cytotoxic or immunosuppressive agent, such as an antitubulin ecules derived from the sera of immunized animals. Useful agent, auristatin, DNA minor groovebinder, DNA replication monoclonal antibodies are homogeneous populations of anti inhibitor, alkylating agent, anthracycline, antibiotic, anti bodies to a particular antigenic determinant of a target anti folate, antimetabolite, chemotherapy sensitizer, cyclooxyge gen. A polyclonal and monoclonal antibody to a target anti nase inhibitor, duocarmycin, etoposide, fluorinated pyrimi gen may be prepared by using any technique known in the art. dine, ionophore, lexitropsin, lipoxygenase inhibitor, 0826 Suitable antibodies include monoclonal antibodies, nitrosourea, platinol, pre-forming compound, purine antime Such as chimeric, humanized, or human antibodies, or an tabolite, puromycin, radiation sensitizer, steroid, taxane, antigen-binding fragment thereof. In one embodiment the antibody fragment is, for example, AC10, BR96, 1F6 or 2F2, topoisomerase inhibitor, Vinca alkaloid, and the like. or growth inhibitory antibody. 0820 Individual cytotoxic agents include, for example, 0827. Individual therapeutic antibodies include, for auristatin (e.g., MMAE, MMAF), azathioprine, bleomycin, example, alemtuzumab (Campath; Leukosite, MA), Allo bortezomib, busulfan, calicheamicin, capecitabine, carbopl mune (BioTransplant), bevacizumab (Avastin, Genetech, atin, carmustine, chlorambucil, cisplatin, cladribine, colas Inc., CA), CEAcide (Immunomedics, NJ), cetuximab (Er pase, cyclophosphamide, cytarabine, dacarbazine, dactino bitux: Imclone Systems Inc., NY), epratuzamab (Immuno mycin, dauorubicin, docetaxel, doxorubicin, duocarmycin, medics, Inc., NJ and Amgen, CA), LymphoCide (Immuno epirubicin, etoposide, etoposide phosphate, fludarabine, fluo medics, Inc., NJ). Oncolym (Techniclone, Inc., CA), OVARE rouracil, fotemustine, ganiclovir, gemcitabine, hydroxyurea, (AltaRex Corporation, MA), Panorex (Glaxo Wellcome, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, NC), rituximab (Rituxan: Genetech), Smart MI95 (Protein mercaptopurine, methotrexate, mitomycin, mitoZantrone, Design Labs, Inc., CA), Smart ID10 (Protein Design Labs, oxaliplatin, paclitaxel, premetrexed, procarbazine, raltitr Inc., CA), trastuzumab (Herceptin: Genetech), and Vitaxin exed, temozolomide, temiposide, thioguanine, thiotepa, (MedImmune, Inc., MD). topotecan, Valganciclovir, vinblastine, Vincristine, Vinore 0828. In certain embodiments the antibodies include, for bine, and maytansine (e.g., DM1, DM4). Drugs that have example, antibodies against the following antigens: tumor been conjugated to the antibody and are currently in clinical associated antigens; antigens associated with pancreatic can trials are auristatin, maytansine, calicheamicin, and duocar cer, malignant disease, autoimmune disease, immune dys mycin (Alley, SC, et al. Current opinion in chemical biology function disease, leukemia, or neurological disease; and 2010, 14, 529-537). antigens against transmembrane activator and CAML-inter 0821. In certain embodiments the cytotoxic agent is dola actor (TACI, Yu et al. Nat. Immunol. 2000, 1, 252-256). statin (e.g., auristatin E, AFP MMAF, MMAE) or derivatives Examples of antigens include: CA125, CA 15-3, CA19-9, L6, thereof. In certain embodiments the cytotoxic agent is a con Lewis Y. Lewis X, alpha fetoprotein, CA 242, placental alka ventional chemotherapeutic, such as doxorubicin, paclitaxel, line phosphatase, prostate specific antigen, prostatic acid melphalan, Vinca alkaloids, methotrexate, mitomycin C, or phosphatase, epidermal growth factor, MAGE-1, MAGE-2, etoposide. In addition, potent agents include CC-1065 ana MAGE-3, MAGE-4, anti-transferrin receptor, p97, MUC1 logs, calicheamicin, maytansine (or DM-1), analogues of KLH, CEA, gp100, MART1, IL-2 receptor, CD4, CD5, CD8, dolastatin 10, rhizoxin, and palytoxin. CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, 0822. In certain embodiments the immunosuppressive CD66a-d, CD67, CD74, CD79a, CD80, CD126, CD138, agent may be, for example, arylcarboxylic derivatives, aza CD154, B7, MUC1, LAL1, HM1.24, HLA-DR, tenascin, thioprine, cyclosporine, cyclooxygenase inhibitors, cyclo VEGF, P1GF, ED-B fibronectin, oncogenes, oncogene prod phosphamide, etanercept, gancyclovir, glucocorticoids or ucts, necrosis antigens, T101, TAG, IL-6, MIF, TRAIL-R1 glucocorticoid analogs, leukotriene receptor antagonists, (DR8), TRAIL-R2 (DR5), human chorionic gonadotropin, 5-lipoxygenase inhibitors, mycophenolate mofetil or meth mucin, P21, MPG, and Neu oncogene product. otrexate, nicotinic acid derivatives, oxicam derivatives, pyra 0829. The antibody can also be a multispecific antibody, Zole-containing derivatives, rapamycin, or tacrolimus. such as a bispecific antibody. Methods for making bispecific 0823. In some preferred embodiments the therapeutic antibodies are known in the art. In some embodiments the agent is a toxin selected from the group consisting of ricin, antibody fragment is an Fv, Fab, Fab', or F(ab'). Other useful abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I, antibodies are heavy chain and light chain dimers of antibod US 2015/001 7246 A1 Jan. 15, 2015 66 ies, single chain antibodies, a minibody, a diabody, a triabody, marketed under the trade name Neulasta (Kinstler O. B. et al. a tetrabody, dsEV, Sc-FV-Fc. (SFV), a fragment produced by a Pharm Res 1996, 13, 996-1002). Fab expression library, an anti-idiotypic (anti-Id) antibody, or 0839. In certain embodiments the therapeutic proteins are multispecific antibodies from antibody fragments. GM-CSF, IFNC-2a, IFNC-2b, IL-2 (Waldmann TA, Nature 0830. In certain embodiments the antibody and proteins Rev. Immuol. 2006, 6, 595-601), erythropoietin (EPO, Mac may comprise one or more radioactive isotopes useful for dougall I C, Curr Hematol Rep 2005, 4, 436-440), growth treating diseased tissue. Suitable therapeutic radionuclides hormone (GH: Zundel M, Peschke B, 2006, WO 2006/ include, but are not limited, iodine-131, iodine-125, bismuth 084888), human growth hormone (hGH; Li CH, Mol Cell 212, bimuth-213, lutetium-177, yttrium-90, yttrium-88, tech Biochem 1982, 46, 31-41), or apomyoglobin (apoMb; Evans netium-99m, copper-62, copper-67, rhenium-188, rhenium SVetal.J. Mol. Biol. 1990,213, 885-897). Other examples of 186, galium-66, galium-67, indium-111, indium-114m, therapeutic proteins and peptides include asparaginase, inter indium-115, boron-10, astatine-211, phosphorus-32, phos ferons (e.g., IFN-O, -3, -y), interleukins, leptin, serum pro phorus-33, Scandium-47, silver-111, praseodyminum-142, teins (e.g., factor VII, factor VIIa, factor VIII, factor IX, and samarium-153, terbium-161, dysprosium-166, holmium factor X), human chorionic gonadotropin (HCG), insulin, 166, rhenium-186, rhenium-188, rhenium-189, lead-212, follicle stimulating hormone (FSH), luteinizing hormone lead-211, radium-223, actinium-225, iron-59, selenium-75, (LH), urate oxidase (uricase), adenosine deaminase (ADA), arsenic-77, strontium-89, molybdenum-99, rhodium-105, and antibody fusion proteins (e.g., tumor necrosis factor palladium-109, praseodymium-143, promethium-149, receptor (TNFR)/Fc domain fusion protein). erbium-169, iridium-194, gold-198, and gold-199. 0840. Other useful proteins include proteins that selec 0831. Additional potential therapeutic radioisotopes tively localize in a particular tissue or region of the body. include carbon-11, nitrogen-13, oxygen-15, bromine-75, Examples of Such proteins include transferrin, HS-glycopro bromine-76, gold-198, actinium-224, iodine-126, iodine tein, coagulation factors, serum proteins, O-glycoprotein, 133, bromine-77, indium-113, ruthenium-95, ruthenium-97, G-CSF, GM-CSF, M-CSF, EPO, and the like. ruthenium-103, ruthenium-105, mercury-107, mercury-203, 0841. In some preferred embodiments M is a derivative of tellurium-121 m, tellurium-122m, tellurium-125m, tellu a therapeutic protein or peptide. For example, fragments of rium-165, tellurium-167, tellurium-168, platinum-197, pal the proteins and chemically modified proteins (e.g., glycosy ladium-109, rhodium-105, praseodymium-142, praseody lation, acylation, amino acid Substitution). In some preferred mium-143, terbium-161, holminum-166, gold-199, cobalt embodiments M is a recombinant protein. 57, cobalt-58, chromium-51, iron-59, selenium-75, thallium 0842. In certain embodiments M is an oligonucleotide, 201, actinium-225, ytterbium-169, and the like. oligonucleotide analog, or small interference RNA (siRNA). 0832. Therapeutic reagents may be attached to the anti Examples of oligonucleotide analogs include peptide nucleic body through partial reduction of the SH group or surface acids (PNAS), locked nucleic acids (LNAs), threose nucleic amines. Therapeutic reagents may also be attached to carbo acid (TNA), and alpha PNAS. hydrate side chains if available. 0843. In certain embodiments the therapeutic agent may 0833. In certain embodiments the therapeutic agents may include one or more copies of the same therapeutic agent or include one or more copies of the same therapeutic agent or combinations of different therapeutic agents. combinations of different therapeutic agents. 0834. In certain embodiments M is a diagnostic agent. EXAMPLES Examples of diagnostic agents include fluorescent probes, 0844. The following examples contain important addi chemiluminescent compounds, radio ligands, mass spectro tional information, exemplification, and guidance that can be metric tags, chromophores, and UV-active compounds. adapted to the practice of this invention in its various embodi 0835. In certain embodiments M is a fluorescent com ments and equivalents thereof. Practice of the invention will pound. Examples of fluorescent compounds include fluores be more fully understood from the following examples, cein, rhodamine, coumarin, green fluorescent protein, which are presented here for illustrative purposes only and BODIPY, Texas Red, Cascade Blue, Lucifer yellow deriva should not be construed as limiting in anyway. tives, phycobiliprotein cyanine dye, lanthanide chelate, and 0845. Instrumentation: quantum dot. Various fluorescent compounds are commer 0846 H-NMR spectra were recorded at 500 MHz cially available (e.g., from Molecular Probes and Invitrogen). (Brukar) and are reported in parts per million (ppm) on the 6 0836. The position at which Mor Mattaches to B can be scale relative to residual CHCl (Ö 7.25) and DMSO-d (Ö a single site or multiple sites. Mor M can be attached to B 2.49). All NMR experiments were performed at room tem through an ester linkage or other crosslinking group. perature (RT) unless otherwise stated. HPLC was performed 0837. In a preferred embodiment M is a therapeutic pro in an Agilent 1100 HPLC system with automatic sample tein or polypeptide. SA macromolecules may conjugate the injector and diode array detector. Analytical HPLC was per protein or peptide at a single specific site or multiple sites. The formed on an XTerraTM C18 column (Waters, 2.5um, 3.0x30 therapeutic proteins may be cytokines, hormones, hemapoi mm). The HPLC method used (method A) was a linear gra etic proteins, blood proteins, enzymes, or peptides. dient of AB solvent (5% B to 95% B in 10 minutes) at a flow 0838. In certain embodiments M is G-CSF, its fragments, rate of 0.6 mL/min. In the case of very hydrophobic com or modified derivatives. G-CSF is a 174-amino-acid glycosy pounds, a Nova-Pack C18 column (Waters, 5 um, 3.9x150 lated cytokine that stimulates the proliferation, survival, and mm) was used with a linear AB gradient (10% B to 95% B in differentiation of neutrophil granulocyte progenitor cells and 10 minutes, then held at 95% B for another 5 minutes) at a mature neutrophils (Hill CP. etal. Proc. Natl. Acad. Sci. 1993, flow rate of 1.0 mL/min (method B). Solvent A was 0.1% 90,5167-5171). G-CSF is rapidly eliminated from the blood. aqueous TFA and solvent B was 0.1% TFA in acetonitrile. Modification of G-CSF using a single SA macromolecule The UV detector was set at 210 nm and 254 nm. Most mass may help stabilize the substance. PEGylated G-CSF has been spectra were collected on a Quadrupole MDS Sciex Q-TRAP US 2015/001 7246 A1 Jan. 15, 2015 67

In a typical experiment, crude or purified samples were dis BDH, Macron, or Mallinckrodt. All other chemicals and solved or diluted in methanol containing 0.1% formic acid reagents were purchased from Alfa Aesar and used as and infused directly into the electrospray inlet. For some large received. molecular weight compounds, mass spectra were collected 0849 Chromatography: on a 4700 Proteomic Analyzer with TOF/TOF optics (AB 0850. Thin-layer chromatography (TLC) analysis was Sciex, Framingham). In a typical experiment, 5uL of sample performed using EMD TLC silica gel 60 F (0.25 mm solution was mixed with 5uI of 1 mg/mL dihydroxybenzoic thickness). The plates were visualized first with UV illumi acid solution in water, then 1 LIL of the mixture was spotted nation, followed by charring with chemical solutions. Differ onto the MALDI plate and air dried. The sample either co ent charring solutions were used: 1) PMA (3% phosphomo crystallized or formed a dried droplet with a matrix. Upon lybdic acid in ethanol w/v); 2) CAM (ceric ammonium laser excitation, the matrix absorbs the laser energy and trans molybdate, 2.5% ammonium molybdate, 1% cerium sulfate fers the energy to the sample, facilitating its ionization and in 10% aqueous sulfuric acid); 3) 5% ninhydrin in ethanol: vaporization. and 4) 2% KMnO, in water. Flash chromatography was per Solvents and Reagents: formed on an ISCO companion using pre-packed columns. 0847 The solvent compositions were on a volume/volume (v/v) 0848 All moisture-sensitive reactions were performed in basis. an inert, dry atmosphere of nitrogen. Reagent grade solvents were used for chromatography and extraction. D-glucamine, Example 1-6 D-glucosamic acid, N-hydroxyphthalimide, and pyridinium p-toluenesulfonate were purchased from TCI America. Selective Protection and Deprotection Strategies for Acetone cyanohydrin, 10% palladium on carbon, 3,4-dihy the Primary and Secondary Alcohol Groups in a dro-2H-pyran, acetic anhydride, formic acid, anhydrous Sugar Alcohol dichloromethane, and anhydrous N,N-dimethylformamide were purchased from Sigma-Aldrich. N-(9-fluorenyl Example 1 methoxycarbonyloxy)succinimide (“Fmoc-OSu’) was pur chased from Chem-Impex International; triethylamine was Synthesis of a Single Free OHSA Molecule Using a purchased from Mallinckrodt; pyridine was purchased from Benzoate Ester and TBDMS Protecting Strategy EMD; and ACS grade solvents were purchased from EMD, 0851

Scheme 12:

TBDMSC1, Imidazole, DMF -7° C. 1/2 hr

O C. during charge Benzoyl chloride Warm to RT pyridine

OH --- TBDMS RT nO OBz OBz I2-a

TBDPSC1, Imidazole, DMF OBZ OBz

US 2015/001 7246 A1 Jan. 15, 2015

0852 Compound I1-a: 34% yield) of starting material was recovered. A total of 0853 Compound I1-a was synthesized based on a similar 0.1045 g (0.175 mmol, 18%) of the tetra-benzoyl diol (I8-a) procedure from the literature (Chem. Pharm. Bull. 2010, 58. was obtained (R, 0.20, confirmed by mass spec). Mass spec 495). 4.55 g (25.0 mmol. 1 eq) of D-mannitol and 5.56 g analysis (electrospray): exact mass 598.18, obtained 599.5 (81.6 mmol. 3.3 eq) of imidazole were partially dissolved in M+H". 20 mL of DMF and cooled to -7°C. In a separate flask, 7.52 0858 Compound I8-a: g (50.2 mmol. 2 eq) of t-butyl-dimethylsilyl chloride was dissolved in 10 mL of DMF. This solution was then added 0859. A solution of 2% iodine in methanol (30 mL) was slowly to the D-mannitol suspension over a period of 20 added to a solution of the crude 12-a (5.50 g. 6.7 mmol) in 15 minutes at -7°C. and the reactionallowed to warm to RT. The mL of THF. The reaction mixture was stirred at RT overnight. DMF was removed under reduced pressure to provide an oil The reaction was quenched with the addition of 3% aqueous that was then dissolved in 90 mL of ethyl acetate. A gummy sodium thiosulfate until the brown color disappeared. The solid formed. The ethyl acetate was decanted away from the Suspension was concentrated in vacuo. The residue was taken solid, which was completely dissolved in 12 mL of water. up in water (-60 mL) and extracted three times with 60 mL of This aqueous phase was extracted with 25 mL of ethyl acetate dichloromethane. The combined organic layers were dried and the ethyl acetate extracts combined. The extracts were over Sodium Sulfate and purified by silica gel column chro then washed three times with 10 mL of water, followed by a matography to obtain 3.84 g of the desired product (96%). wash with 15 mL of brine. The organic phase was dried over TLC (EtOAc:hexanes=1:1. Rf-0.5). Mass spec analysis anhydrous NaSO4, filtered, and concentrated under reduced (electrospray): exact mass 598.2, obtained 621 M--Na" and pressure. The resulting material was purified by silica gel 1219 (2M+Na". column chromatography to obtain 2.10 g of desired product 0860 Compound I3-a (Converting from I8-a to I3-a): (R-020, EtOAc:hexanes–25:75, charring by 2% KMnO,). 0861. A solution oft-butyldimethylsilyl chloride (256 mg, MALDI: exact mass 410.25, obtained 433.44 M+Na". 1.0 equiv.) in anhydrous dichloromethane (5 mL) was added 'H-NMR (500 MHz, DMSO-d): 8 4.32 (d. J=5.7 Hz, 2H), dropwise via Syringe pump at a rate of 2.5 mL/h to a stirred 4.02 (d. J=7.1 Hz, 2H), 3.76 (dd, J=10.4, 3.0 Hz, 2H), 3.57 solution of compound k (10g, 1.7 mmol) and imidazole (116 3.48 (m, 4H), 3.43 (dtd, J=8.7, 5.9, 3.0 Hz, 2H), 0.83 (s, 18H), mg, 1 equiv.) in anhydrous DMF (10 mL) under nitrogen at 0.00 (s, 12H). Higher substituted byproducts were also RT. The reaction was then stirred for an additional hour. Next, present (confirmed by mass spec): 5.05 g of tri-TBDMS the reaction was placed in an ice bath and quenched with 70 protection (39% yield) (R-0.53) and tetra-TBDMS substitu mL of water added in small portions. The mixture was tion (R-0.71). extracted three times with 40 mL of dichloromethane. The 0854 Compound I2-a: combined organic layers were washed three times with 20 mL 0855 0.9746 g (2.374 mmol, 1 eq) of I1-a were dissolved of water, followed by two washes with 20 mL of brine. The in 4.6 mL (57 mmol) of pyridine and cooled to 0°C. Next, organic layer was dried over Sodium Sulfate overnight, fil 1.50 mL (12.2 mmol. 5.1 eq) of benzoyl chloride was added tered, and concentrated in vacuo at 40°C. The residue was drop wise. The reaction was allowed to warm to RT and stir purified by silica gel column chromatography to obtain the overnight. The majority of the pyridine was removed by desired product (584 mg. 48%). TLC (EtOAc:hexanes=2:3. evaporation under reduced pressure, and the resulting residue 254 nm UV): Rf 0.83. Mass spec analysis (electrospray): was dissolved in 15 mL of ethyl acetate. The ethyl acetate was exact mass 712.27, obtained 592 M-OBZ", 713 M+H". then washed with 5 mL of 1 NHCl (aq), followed by 5 mL of 'H-NMR (500 MHz, chloroform-d): 8 8.07-7.99 (m, 4H), saturated NaHCO (aq). The ethyl acetate was dried over 8.00 (dd, J=8.2, 1.4 Hz, 2H), 7.92 (dd, J=8.4, 1.3 Hz, 2H), anhydrous NaSO, filtered and concentrated under reduced 7.64-7.32 (m, 12H), 6.16 (dd, J=6.9, 2.5 Hz, 1H), 6.08 (dd. pressure, and purified by silica gel column chromatography to J=8.2, 2.6 Hz, 1H), 5.56 (dt, J–7.0, 4.6 Hz, 1H), 5.35 (dt, obtain 1.59 g of the desired product (80% yield). Mass spec J=8.1, 3.3 Hz, 1H), 4.04-3.94 (m, 2H), 3.89 (dd, J=11.3, 4.8 analysis (electrospray): exact mass 826.36, obtained 827.7 HZ, 1H), 3.79 (dd, J=13.3, 3.8 Hz, 1H), 2.50 (s, 1H), 0.81 (s, M+H"; 849.7 M+Na". 9H), -0.02 (s.3H), -0.06 (s, 3H). 0856 Compound I3-a: 0857 0.1060 gofelemental iodine was dissolved in 10 mL Example 2 of methanol to create an approximately 1% by weight solu tion. Next, 0.800 g (0.967 mmol. 1 eq) of I2-a was dissolved Synthesis of a Single Free OHSA Molecule Using in 7.5 mL of the I/MeoH solution at RT. An additional 1.0 the Acetate Ester and TBDMS Protecting Strategy mL of THF was added to obtain a homogeneous solution. The reaction was monitored by TLC. After 9 hours, the reaction 0862 was quenched by the drop wise addition of 3% NaSO (aq) until all of the iodine color was consumed. MeOH and THF were removed under reduced pressure and the residue dis Scheme 13: solved in 6 mL of ethyl acetate and 2 mL of water. The phases Acetic were separated and the organic phase washed twice with 2 mL anhydride of water, followed by 2 mL of brine. Ethyl acetate was yridine removed by evaporation under reduced pressure. The result ing material was purified by silica gel column chromatogra phy to obtain 0.2338g of the desired product (0.328 mmol. 34% yield, R-0.57, 25.75 EtOAc:hexanes). Mass spec analysis (electrospray): exact mass 712.27, obtained 713.6 M+H"; 735.3 M+Na". A total of 0.2681 g (0.324 mmol, US 2015/001 7246 A1 Jan. 15, 2015 69

-continued Example 3 OAc THP (Tetrahydropyranyl) and TBDMS Protecting Strategy 0867

Scheme 14:

Dihydro pyran -e- PPTS, CHCI or Amberlyst 15, CHCl2 or bis(trimethylsilyl sulfate, CH2Cl2

OTHP TBDMSO 0863 Compound I2-b: 0864 Acetic anhydride (7 mL, 5.1 equiv.) was added dropwise via Syringe to a stirred solution of compound I1-a (4.3 g, 10.5 mmol) in pyridine (10 mL) at 0°C. The reaction was allowed to warm to RT overnight. The reaction was 0868 Compound I4-a: quenched by the addition of water in small portions (-20 mL 0869. Three different methods were explored for THP pro total) to produce a biphasic liquid, which was extracted three tection of the primary hydroxyl group using I3-a as the start times with 40 mL of ethyl acetate. The combined organic ing material. Method A: 29.2 mg of I3-a (41 umol) were layers were washed three times with 15 mL of 0.1 NHCl, dissolved in 100 uL of CHCl in an HPLC vial, followed by the addition of 4.17 uL of 3,4-dihydro-2H-pyran and 20 L of followed by 20 mL of brine, and then dried over sodium 0.1 mg/LL beads in CHC1. The mixtures were stirred at RT sulfate overnight. The dried organic layer was filtered and for 2 hrs. TLC indicated that some starting material remained concentrated in vacuountil no UV absorption was observed at (Rf 0.28 for starting material), and a new spot with Rf-0.59 254 nm on the TLC plate, Suggesting the removal of residual was observed. Additional starting material (4.17 uL) was pyridine, to yield the product (5.9 g, 97%), which was used added. After 30 minutes the reaction was complete with a without further purification. TLC (EtOAc:hexanes=1:3, char single spot detected by UV, which is presumably the product. ring by 2% KMnO): Rf=0.65. The mixture was filtered through glass wool to obtain the crude product. Method B: 14.7 mg of I3-a (20 umol) was dissolved in 100 uL of CHCl in an HPLC vial, followed by 0865 Compound I3-b: the addition of 1.92 uL of 3,4-dihydro-2H-pyran and 1.1 LL of 50 mMbis(trimethylsilyl)sulfate in CHC1. The mixtures 0866 Compound I2-b (4.9 g, 8.7 mmol) was suspended in were stirred at 0° C. for 2 hrs. TLC indicated that some starting material remained (Rf 0.28), and a new spot with methanol (18 mL) by sonication for 5 minutes. A solution of Rf 0.59 was observed. Additional starting material (1.92 uL) 2% (w/v) iodine in methanol (17 mL) was added, and the was added. After 30 minutes, approximately 50% of the start reaction was stirred at RT overnight. The reaction was ing material remained in the Solution. Method C, 21.4 mg of quenched by the addition of 3% aqueous sodium thiosulfate I3-a (30 umol) was dissolved in 100 uL of CHCl in an in small portions until no brown color remained (~15 mL HPLC vial, followed by the addition of 3.82 uL of 3,4-dihy total). The Suspension was concentrated in vacuo to remove dro-2H-pyran (1.5 equiv.) and 10LL of 76 mg/mL pyridinium most of the MeOH, diluted by the addition of 15 mL water, p-tolenesulfonate (PPTS) in CHC1. The mixtures were then extracted three times with 30 mL of dichloromethane. stirred at RT for 2 hrs. TLC indicated that some starting The combined organic layers were washed with 20 mL of material remained (Rf 0.28), and a new spot with Rf-0.59 was observed. Additional starting material (3.82 uL) was brine, dried over sodium sulfate, and purified by silica gel added. After 30 minutes the reaction was complete. 150 uL of column chromatography to obtain the desired product (720 ethyl ether (PPTS forms white precipitate) was added to the mg, 18%). TLC (EtOAc:hexanes=1:1, charring by 2% vial plus 100 uL of 4M NaCl. The mixture was extracted KMnO): Rf-0.57. Mass spec analysis (electrospray): exact twice with CHC1. The organic layers were combined to mass 464.21, obtained 333 M-OTBDMSI: 405 M-OAc)", obtain the crude product. Mass spec analysis (electrospray): 465 M+H", 483 M+HOI",951 2M--Nal". exact mass for I4-a 796.33 and observed 797.8 M+H". US 2015/001 7246 A1 Jan. 15, 2015 70

Example 4 Example 5 Selective Protection of a Single Primary Alcohol of Benzyl Protection of OH SA Using the TBDPSC1 Strategy 0873 0870

Scheme 16: Scheme 15: OH TBDPSCI, Imidazole, DMF -7 C. degrees, 1/2 hr 1) NaH, DMF at 0°C. OH -- 2) BnBr at 0°C. HO O -e- Warm to RT, 1/2 hr O

I8-d OB

OH OH I34-a Major HO O

O w

OTBDPS I5-d

0874 Compound I5-d: I1-d Minor 0875. A solution of 1.0607 g (6.54 mmol. 1.0 eq) 2.3-O- isopropylidine-D-threitol (TCI America) dissolved in 2 mL 0871 Compound I34-a: DMF was added to a suspension of 0.2959 g of NaH (7.40 0872 0.53 g (6.4 mmol) of D-mannitol and 0.54 g (7.9 mmol. 1.13 eq.) as a 60% by wt. dispersion in oil in 4 mL of mmol) of imidazole were dissolved by heating in 16 mL of DMF at 0°C. The addition occurred over a period of 10 DMF, then cooled to -7°C. In a second flask, 1.65 mL (6.4 minutes, followed by 30 minutes of additional reaction time. mmol) of t-butyl-diphenylsilyl chloride was dissolved in 10 To this solution, 0.780 mL (6.56 mmol. 1.0 eq) of benzyl mL of DMF. This solution was then added to the mannitol bromide was added slowly and the resulting mixture stirred solution over a period of 20 minutes at -7°C. and stirred for for 20 minutes at 0°C. TLC analysis (40:60 ethyl acetate: an additional 5 minutes. The DMF was removed under hexane on silica, CAM stain) indicated consumption of the reduced pressure to provide an oil that was then dissolved in starting material and the product with R, 0.28. The majority 50 mL of ethyl acetate. A white solid (imidazole hydrochlo of the DMF was removed by evaporation under reduced pres ride) precipitated and was removed by filtration. The ethyl sure. The resulting oil was dissolved in 5 mL of water and acetate extract was washed twice with 10 mL of water, fol extracted three times with 15 mL of ethyl acetate. The organic lowed by a wash with 10 mL of brine. The organic phase was extracts were combined, washed with 5 mL of brine, dried concentrated under reduced pressure, providing 2.4 g of oil. over anhydrous NaSO filtered, and concentrated under Analysis by TLC indicated that the majority of the product reduced pressure. The resulting residue was then purified by was mono-protected (I34-a). Mass spec analysis (MALDI): silica gel column chromatography to obtain 0.997 g (3.95 exact mass 420.20, observed 443.39 (M+Na" (R-021, mmol, 60.4% yield) of the desired product. Mass spec analy 25:75 EtOAc:hexanes) and a minor spot corresponding to the sis (MALDI): exact mass 252.14 Da, observed 275.95 di-protected substance (R, 0.93). Mass spec analysis M+Na". HPLC showed one peak at 4.387 minutes for the (MALDI): exact mass 658.31, observed 680.7 M+Na". purified product (method A). US 2015/001 7246 A1 Jan. 15, 2015 71

SI ovo

:LIQUIQUIOS

US 2015/001 7246 A1 Jan. 15, 2015 73

Example 6 monitored by TLC or HPLC: the amide coupling is normally complete within 1-4 hrs. After the reaction, the solution is Synthesis of Monofunctional SA Crosslinking passed through a C18 column to desalt (if sufficiently pure) or Reagent p3-4f subjected directly to a preparative HPLC purification using a 0876 Compound I37-b: waterfacetonitrile gradient system. The collected fractions 0877. Diisopropyl azodicarboxylate (DIAD) (178 uL, are combined and lyophilized to obtain the product. The 0.906 mmol. 1.2 equiv.) was added dropwise via syringe to a crude product can also be purified by silica gel chromatogra solution of compound I3-b (350 mg, 0.753 mmol) and triph phy (usually over 90% yield, if HPLC is employed the recov enylphosphine (238 mg, 0.906 mmol. 1.2 equiv.) in dry tet ery is lower). The identity of the product is checked by mass rahydrofuran (21 mL) under nitrogen at 0°C. After approxi spectrometry and the structure confirmed by 'HNMR. mately one hour of stirring at 0° C. in the dark, diphenylphosphoryl azide (196 uL, 0.906 mmol. 1.2 equiv.) Example 7 was added dropwise via Syringe. The reaction was warmed to RT and stirred in the dark for 3 days. The reaction mixture was Synthesis of Monofunctional SA Crosslinking concentrated in vacuo and purified by Silica gel column chro Reagent p3-4b matography to obtain the desired product (0.294 g. 80% yield). TLC (EtOAc:hexanes=1:3, charring by PMA): Rf 0. 0888 Compound p3-4b: 52. Mass spec analysis (electrospray): exact mass 489.21, 0889 Compound p3-4b is prepared following a similar observed 490 M+H"; 512 M+Na"; 1001 2M--Na". protocol as compound p3-4f. First, the NHS ester is synthe 0878 Compound I38-b: sized by following a similar protocol as trifluoromethylben 0879 Triphenylphosphine (470 mg, 1.80 mmol. 3 equiv.) Zoic acid. 3-maleimidopropionic acid (1.1 equiv. of com and deionized water (100 uL) were added to a solution of pound I4-ba), N,N'-dicyclohexylcarbodiimide (DCC, 1.1 compound I37-b (294 mg. 0.600 mmol) in THF (10 mL). The equiv.), and N-hydroxysuccinimide (1.1 equiv.) in DCM is reaction was stirred at RT overnight and then the mixture added to a flask. Ice/water may be used if the scale of the concentrated in vacuo. Toluene (~5 mL) was added and the reaction is over a few grams. After stirring the reaction for a mixture concentrated again in vacuo. The residue was puri few hours, a white solid precipitated out. After filtration, the fied by silica gel column chromatography to obtain the prod organic solvent is evaporated under low pressure to produce uct (91 mg, 33%). TLC (EtOAc:hexanes=3:1, charring by an oil. The oil is added directly to the KCO solution con Ninhydrin): Rf 0.17. Mass spec analysis (electrospray): taining p3-4 following the same protocol as compound exact mass 463.22, obtained 464 M+H"; 486 M+Na";949 p3-4f. The product is purified by HPLC or silica gel chroma 2M--Na". tography (usually over 90% yield, if HPLC is employed the 0880 Compound I5-bb: recovery is lower). The identity of the product is checked by 0881 TBDMS of I38-b is deprotected following a similar mass spectrometry and the structure confirmed by HNMR. protocol as converting I2-a to I8-a using a 2% iodine in methanol solution. The crude product can be either purified Example 8 by silica gel column chromatography or used directly for the next reaction if it is sufficiently pure (>90%). 0882 NHS Ester of Trifluoromethyl Benzoic Acid: Synthesis of Monofunctional SA Crosslinking 0883 20 mg (0.0869 mmole) of 4-(1-azi-2.2.2-trifluorom Reagent p3-4e ethyl)benzoic acid, 21.6 mg (0.1052 mmole) of DCC, 11 mg Dithiopyridine Propionic Acid: (0.0956 mmole) of N-hydroxysuccimide, and 435 uL of dim 0890 ethylformamide (DMF) was added to a 1.5 mL ultracentri 0891) 2,2-dithiodipyridine in methanol (~1M solution) fuge tube. The mixture was vortexed for 30 seconds and then purged with N2 is added to an oven-dried three-necked flask nutated at RT. HPLC indicated the completion of the reaction under N2 and equipped with a stir bar at RT. 3-mercaptopro within an hour (starting material: to 3.73 minutes; NHS pionic acid is added dropwise to this mixture. The solution ester: to 3.99 minutes). The crude reaction mixture was used turns yellow and is allowed to stir for 2-4 hrs. The solvent is directly for the next step without purification. HPLC condi removed in vacuo and the residue purified by Silica gel col tions: XterraTMRP18 column, 5 nm, 4.6x250 mm; Buffer A: umn chromatography to obtain the desired product (usually 0.1% TFA in water; Buffer B: 0.1% TFA in acetonitrile; quantitative yield). The identity of the product is checked by Eluted with isocratic 60% B. mass spectrometry and the structure confirmed by HNMR. 0884 Compound p3-4: (0892 NHS Ester of Dithiopyridine Propionic Acid: 0885 KCO solution (~1 M) in water is added to a flask 0893 NHS ester is synthesized following a similar proto containing compound I5-bb. If the compound is not com colas trifluoromethylbenzoic acid and dichloromethane used pletely soluble, a small amount of MeOH can be added. The progress of the reaction is monitored by TLC (charring by as a solvent. The progress of the reaction is monitored by PMA). The reaction is normally done within 1 hr to obtain HPLC or TLC. After the reaction, the solid precipitate is compound p3-4, which is used directly for the next reaction filtered and the solvent removed in vacuo. The residue oil is without work up and purification. used directly for the next reaction. 0886 Compound p3-4f: 0894 Compound p3-4e: 0887. The reaction mixture of the NHS ester of trifluorom 0895. The compound is synthesized as described for p3-4f ethylbenzoic acid is filtered and added directly into KCO using the NHS ester of dithiopyridine propionic acid and Solution containing compound p3-4 (~ 1:1 molar ratio). The p3-4 as the starting materials. The identity of the product is pH of the solution is checked. The optimal pH for this reaction checked by mass spectrometry and the structure confirmed by is between pH 8.0 and 9.0. The progress of the reaction is HNMR. US 2015/001 7246 A1 Jan. 15, 2015 74

Example 9 Synthesis of Monofunctional SA Crosslinking Reagent p3-4a 0896

Scheme 22:

N-hydroxyphthalimide OH -- TBDMSN P(Ph)/DIAD

Hydrazine/pyridine? acetic acid (1:32:16)

OBZ OBz

". NaOH in MeOH

0897 Compound I36-b: (0901) Compound I5-aa: 0902 Compound I5-aa is prepared as described for con 0898 Diisopropyl azodicarboxylate (29 uI) was added Verting I2-a to I8-a using a 2% iodine in methanol solution. dropwise via Syringe to a solution of compound I3-a (70 mg. The crude product can be either purified by silica gel column 0.098 mmol), N-hydroxyphthalimide (25 mg 0.15 mmol. 1.5 chromatography or used directly for the next reaction if it is equiv.), and triphenylphosphine (39 mg, 0.15 mmol. 1.5 sufficiently pure (>90%). equiv.) in THF (ACS grade, 1 mL) in an ice bath. The reaction (0903 Compound p3-4a: was allowed to warm to RT overnight. The starting material 0904 Compound I5-aa (~50 mg) is added to a 10 mL was mostly consumed. The mixture was concentrated in reaction vial, followed by 1% NaOH in MeOH solution. The vacuo and purified by silica gel column chromatography to reaction mixture is stirred at RT and the progress of the obtain the desired product (71 mg, 84%). The ESI-MS m/z. reaction checked by TLC (UV and PMA charring). After was consistent with M-OBZ" and M+H" (736 and 858, completion, the MeOH is removed in vacuo. DCM is added respectively). into the residue solid. After mixing for a few minutes, the (0899 Compound I36-bb: DCM layer is dried in vacuo to obtain the product. If the DCM layer contained some benzoic acid, the residue is redissolved 0900 Compound I36-b (70 mg, 0.082 mmol) was mixed in water and acidified to pH 2-3 using diluted HC1. The with 500 uL of a hydrazine:pyridine:acetic acid (1:32:16, aqueous solution is extracted with DCM to eliminate the Volume by Volume) solution by Vortexing. After approxi residual benzoic acid. The aqueous Solution is adjusted to pH mately 10 minutes, the reaction was quenched with 10 mL of 8-10 using diluted NaOH. The aqueous solution is extracted water, which resulted in an initial white turbidity, but then all three times with DCM. The DCM layers were combined and dried to obtain the product. The identity of the product is turbidity dissolved. The reaction was extracted three times checked by mass spec and the structure confirmed by with 3 mL of dichloromethane. The crude combined organic H-NMR. phase was analyzed by mass spec (electrospray): exact mass (0905 Compound I5-b: 727.28, observed 606 M-OBz"; 728 M+H"; 1456 (2M+ 0906 A 2% solution of iodine in methanol (4 mL) was H'. An alternative method using tert-butyl N-hydroxycar added to a solution of compound I4-ba (252 mg, 0.515 mmol) bamate (BocN-OH) was also tested on a very small scale and in methanol (2 mL). The reaction was stirred at RT overnight. resulted in the desired product as confirmed by mass spec. The reaction was quenched by dropwise addition of 3% aque US 2015/001 7246 A1 Jan. 15, 2015

ous sodium thiosulfate until no brown color remained. The -continued mixture was concentrated in vacuo, taken up in water (~20 OH OH mL), and extracted three times with 20 mL of dichlo romethane. The combined organic layers were dried over Sodium sulfate overnight, filtered, concentrated, and dried under vacuum to yield the product (176 mg.91%), which was OH used for the next reaction without further purification. TLC (EtOAc:hexanes=3:2, charring by PMA): Rf 0.47. The ESI MS m/z was consistent with M+H", 376; M+Na", 398: and 2M--Na', 773. (0908 Compound I9-a: Example 10 (0909. A solution of DIAD (49 uL, 3.0 equiv.) in THF (0.1 mL) was added slowly via Syringe to a solution of compound Synthesis of Homobifunctional SA Crosslinking k (49.5 mg 0.083 mmol), N-hydroxyphthalimide (40.5 mg. Reagent p6-4aa 3.0 equiv.), and triphenylphosphine (65 mg, 3.0 equiv.) in 0907 THF (0.8 mL) in an ice bath under nitrogen. The solution quickly turned bright orange. After approximately 30 min utes, the reaction mixture was allowed to warm to RT and the Scheme 19: orange color faded. The reaction was stirred overnight. The volatiles were removed in vacuo, and the residue was purified on a silica gel column to yield the product (31 mg, 42%) with N-hydroxyphthalimide OH --> acceptable purity (about 85%). HPLC (TFA05 method, P(Ph)/DLAD THF detected by UV absorbance at 220 nm) retention time=9.20 min. Mass spec analysis (electrospray): exact mass 888.22 Da, obtained 766 M-OBZ"; 911 M--Na". 0910 Compound p6-4aa: 0911 Phthalimide protecting groups and the BZ groups are removed and the product purified following the protocol described in example 9 to obtain compound p6-4aa. Example 11

1)NH2NH2/pyridine/AcOH Synthesis of Homobifunctional SA Crosslinking 2) 1% NaOH MeOH Reagent p6-4bb 0912

Scheme 20:

O O OH H N Solution

Y US 2015/001 7246 A1 Jan. 15, 2015 76

-continued CF

NYCF, N

(0913 Compound I9-bb: Example 12 0914 Diazide compound I9-bb is converted from pro tected di-hydroxyl mannitol (I8-b) following the same proto Synthesis of SA Crosslinking Reagent p6-4ff colas described for monoazide compound 37-b in example 6. The compound is purified by Silica gel chromatography. The 0921 Compound p6-4ff. identity of the product is checked by mass spec and the 0922 Homobifunctional maleimide compound p6-4ff is structure confirmed by H-NMR. The purity is checked by synthesized from p6-4 following the same protocol as TLC with charring (PMA). described for monofunctional maleimide compound p3-4f in (0915 Compound I9-bc: example 6. The compound is purified by silica gel chroma 0916 Diamine compound I9-bc is converted from the tography. The identity of the product is checked by mass spec diazide compound I9-bb following the same protocol as and the structure confirmed by 'H-NMR. The purity is described for monoamine compound 38-b in example 6. The checked by TLC or HPLC. compound is purified by silica gel chromatography. The iden Example 13 tity of the product is checked by mass spec and the structure confirmed by H-NMR. The purity is checked by TLC with Synthesis of SA Crosslinking Reagent p6-444 charring (PMA). (0917 Compound p6-4: 0923 Compound p6-4ee: 0918. The deprotection of the acetate group of diamine 0924 Homobifunctional maleimide compound p6-4ee is compound I9-bc is performed following the same protocolas synthesized from p6-4 following the same protocol as described for compound p3-4 from I5-bb in example 6. After described for monofunctional maleimide compound p3-4e in the completion of the deprotection, the mixture is used example 8. The compound is purified by silica gel chroma directly to carry out the next step. tography. The identity of the product is checked by mass spec (0919 Compound p6-4bb: and the structure confirmed by 'H-NMR. The purity is 0920 Homobifunctional maleimide compound p6-4bb is checked by TLC or HPLC. synthesized from p6-4 following the same protocol described for monofunctional maleimide compound p3-4b in Example 14 example 7. The compound is purified by silica gel chroma tography. The identity of the product is checked by mass spec Synthesis of Heterobifunctional Thrietol and the structure confirmed by 'H-NMR. The purity is Crosslinking Reagent p2-2ab checked by TLC or HPLC. 0925 Scheme 18:

O MsCL/EtN O Boc-N-OH O ... I III -e- III DBU III O O \ O O-Bn O-Bn O-Bn

HO MSO BocNocHN No. I3-d I4-da I4-db 1, 4-cyclohexadiene Pd(C US 2015/001 7246 A1 Jan. 15, 2015 77

-continued

P(Ph)3, THF, DPPA O O O NH2 N DIAD OH

BochNNo BocHN- O BochNNo I6-db I6-da I5-da FATHF/H2O HO HO O O

-- HO ". pH > 8.0 --HO ". NH2 ---> NH N O TFAHN- O O O TFAHN- O p1-2a N O

O O

0926 Compound I4-da: added via syringe. The mixture was stirred at RT. Slow con 0927 Methanesulfonyl chloride (75 uL) was added drop version was observed, and additional excess 1,4-cyclohexa wise via a syringe to a stirred solution of compound I3-d (195 diene was added periodically. The reaction was not complete mg, 0.773 mmol) and triethylamine (300 uL, 2.8 equiv.) in after one day of stirring; therefore, formic acid (50 uL) was dichloromethane (2 mL) in an ice bath. The reaction was used as an alternative hydrogen transferreagent. After stirring stirred in the ice bath for one hour, then quenched with 2 M for 2 days the reaction was complete. The mixture was care aqueous NHCl (~2 mL). The organic phase was separated fully filtered through a Celite pad and concentrated to obtain out and washed again with 2 Maqueous NHCl (-2 mL). The the product (25 mg, 83%), which was used for the next step combined aqueous layers were back-extracted three times without further purification. TLC (EtOAc:hexanes=2:3, visu with 3 mL of dichloromethane. The combined organic phase alized by CAM charring): Rf 0.22. Mass spec analysis (elec was washed with 2 mL of brine, dried over sodium sulfate, trospray): exact mass 277.15 Da, observed 278 M+H"; 300 filtered, and concentrated to obtain the crude product (quan M+Na"; 555 2M-H"; 577 (2M+Na". titative yield assumed) without further purification. TLC 0932 Compound I6-da: (EtOAc:hexanes=2:3, visualized by 254 nm UV or PMA 0933. The primary OH group in I5-da is converted to azide charring): Rf-0.46. following the same protocol described for compound I37-bin 0928 Compound I4-db: example 6. The compound is purified by silica gel chroma 0929. A solution of compound I4-da (255 mg, 0.773 tography. The identity of the product is checked by mass spec mmol) in diethyl ether (1 mL) was added via Syringe to a and the structure confirmed by 'H-NMR. The purity is stirred solution of DBU (175 uL. 1.5 equiv. with respect to checked by TLC with charring (PMA). compound I4-da) and N-Boc-hydroxylamine (145 mg, 1.4 0934 Compound I6-db: equiv.) in diethyl ether (1 mL) in an ice bath under nitrogen. 0935 The azide group in I6-da is reduced to an amine The reaction mixture was stirred at RT for 3 days. Slow following the same protocolas described for compound I38-b conversion was observed, though the reaction profile was in example 6. The compound is purified by silica gel chroma clean. The ether was evaporated and the reaction mixture tography. The identity of the product is checked by mass spec re-dissolved in acetonitrile (2 mL) and heated to reflux over and the structure confirmed by 'H-NMR. The purity is night. Faster conversion was observed, though the reaction checked by TLC with charring (PMA). profile became significantly more complicated. The reaction 0936 Compound p1-2aj: mixture was concentrated and purified via silica gel chroma 0937. The acetonide (isopropylidene ketal) protection tography to obtain the product (48 mg, 17% over two steps). group and the Boc were simultaneously deprotected follow TLC (EtOAc:hexanes=2:3, visualized by 254 nm UV or ing the procedure in the literature (Leblanc, Y. et al. J. Org. CAM charring): Rf 0.75. Mass spec analysis (electrospray): Chem. 1986, 51,789). Trifluoroacetic acid (0.2M) is added to exact mass 367.20 Da, observed 368 M+H"; 390 M+Na"; a stirred solution of compound I6-db in 4:1 THF/water at 0° 757 2M--Nal". C. The resulting solution is allowed to warm to RT and left 0930 Compound I5-da: overnight. The reaction is neutralized by the addition of con 0931. A 10-mL pear-shaped flask equipped with a stir bar centrated ammonium hydroxide, and THF removed under was charged with 100 mg of 10% palladium on carbon, then reduced pressure to obtain the product. purged twice with nitrogen. A solution of compound I4-db 0938 Compound p1-2ab: (40 mg, 0.109 mmol) and 1,4-cyclohexadiene (200 uL. 0939. The amine group of p1-2aj is coupled to the NHS approximately 20 equiv.) in 1:1 THF/MeOH (1 mL) was ester of 3-maleimido propionic acid following the same pro US 2015/001 7246 A1 Jan. 15, 2015 78 tocol as described for compound p3-4b in example 7. The compound is purified by silica gel chromatography. The iden tity of the product is checked by mass spec and the structure confirmed by 'H-NMR. The purity is checked by TLC or HPLC.

Scheme 22:

2% I2 in MeOH TBDMSO

MsC1, TEA DCM, 0 degrees

Boc-N-OHDBU -- MISO

TFATHF/H2O

OH

o TFADCM

US 2015/001 7246 A1 Jan. 15, 2015 79

Example 15 0947 Compound I4-ad: Synthesis of Heterobifunctional Thrietol 0948. A solution of carbon tetrabromide (300 mg, “may Crosslinking Reagent p2-2ab contain up to ca. 6% water,” approximately 1.05 equiv.) in 1 mL dichloromethane was added to a solution of compound (0940 Compound I5-b: I3-a (505 mg, 0.84 mmol) indichloromethane (3 mL) in an ice 0941. A 2% solution of iodine in methanol (4 mL) was bath. A solution of triphenylphosphine (275 mg, 1.25 equiv.) added to a solution of compound I37-b (252 mg 0.515 mmol) in 1 mL dichloromethane was added. The reaction was stirred in methanol (2 mL). The reaction was stirred at RT overnight. The reaction was quenched by the dropwise addition of 3% in an ice bath for approximately 2 hours, and then warmed to aqueous sodium thiosulfate until no brown color remained. RT and stirred overnight. TLC indicated a trace amount of The mixture was concentrated in vacuo, taken up in water starting material remained, but the reaction profile was rather (~20 mL), and extracted three times with 20 mL of dichlo dirty. The reaction mixture was concentrated in vacuo and romethane. The combined organic layers were dried over purified by silica gel column chromatography to obtain the Sodium sulfate overnight, filtered, concentrated, and dried product (209 mg, 37%). TLC (EtOAc:hexanes=1:1, visual under vacuum to obtain the product (176 mg, 91%), which ized by UV 254 nm): Rf 0.82. The ESI-MS m/z was consis was used for the next reaction without further purification. tent with M-OBZ",539,541; M-Br",581; M-OH, 643, TLC (EtOAc:hexanes=3:2, charring by PMA): Rf 0.47. 645; M+H, 661, 663; M+Na, 683 and 685; and 2M-- Mass spec analysis (electrospray): exact mass 375.13 Da, Na, 1344, 1346, and 1348. obtained: 376 M+H"; 398 M+Na"; 773 (2M+Na". (0942 Compound I5-bb and 15-bc: Example 17 (0943. The OH group of 15-b is converted to the Boc protected aminooxy following the same protocol described for compound I4-db in example 11. The compound is purified Alkylation of an OH Group by Silica gel chromatography. The identity of the product is checked by mass spec and the structure confirmed by 0949 H-NMR. The purity is checked by TLC or HPLC. 0944 Compound p1-4a: 0945. The acetate of 15-bc is deprotected in KCO solu Scheme 24: tion following the protocol described for compound p3-4 in example 6. After deprotection, the solution is neutralized and MeIAgO/EtO extracted with DCM. The DCM layers were evaporated in He vacuo and purified by silica gel chromatography to obtain compound ol-a. The Boc of ol-a is deprotected following the OR protocol described for compound p1-2aj. After evaporation of C18H42O6Si2 the solvent, a pure product is obtained. The identity of the Exact Mass: 410.25 product is checked by mass spec and the structure confirmed MOI. Wit.: 410.69 by 'H-NMR. The purity is checked by TLC or HPLC. I1-b

Example 16 Synthesis of Br-Substituted SA 0946 OR OR Scheme 23: C22H50O6Si2 Exact Mass: 466.31 MOI. Wit.: 466.80 O1-a R: one of them is TBDMS, and the rest of them are H OBZ OBZ C40H44O10Si Exact Mass: 712.27 MOI. Wit.: 712.86 0950 Compound O1-a: I3-a 0951 Methyl iodide (1.3 mL) was added to a stirred mix ture of the starting material, I1-b (100 mg), silver (I) oxide (1.97g), and crushed 4 AMS (500 mg) in diethyl ether (5 mL). The reaction was heated at reflux for 8 hr, then cooled to RT and stirred for 2 days. TLC indicated complete conver OBz OBz sion, and ESI-MS data suggested the formation of the C40H43O9Si. expected product: 467 M+H, 489 M+Nal", 956 2M-- Exact Mass: 774.19 Na". Note: in this reaction, the starting material was a Mol. Wit.: 775.75 byproduct of the silylation reaction of mannitol (Scheme 12). Il-b is a regional isomer of I1 -a, as it has the same mass spec data but a lower Rf value because one of the TBDMS is coupled to the secondary OH group of the mannitol. US 2015/001 7246 A1 Jan. 15, 2015

Example 18 Dimerization and Tetramerization of Threitol 0952

Scheme 24: OB OB OB \/ CH2Cl2. O C. O NaO O -- Fo1 No O -o- O O Warm O O to OOl OB temp generate in situ generated in situ I17-a I4-dic I16-a C28H38O-7 Exact Mass: 486.26 MOI. Wit.: 486.60

Po?C 10% wtfwt THF, MeOH Excess

OB

OB S(\ KrO O / O. O OBn SK DMF, X Y-N \ "K O. O. O CH2Cl2. I1.8-b ONa O -78 C. O O a- In situ generation X O O p-A Warm in DMF OH Y ( O OOlto FC O 1 C2H32O7 O. O. temp N --O Exact Mass: 396.21 X I MOI. Wt: 396.47 OB N O X, I18-a O C43H66O13 Exact Mass: 790.45 >No O MOI. Wt: 790.98 BO In situ generation in CH2C2 I20-a

0953 Compound I17-a: evaporation under reduced pressure. The resulting oil was 0954 0.1223 g (0.485 mmol. 1 eq) of I3-d was dissolved dissolved in 5 mL of water and extracted three times with 15 in 2.1 mL of CHC1, with 0.075 mL (0.538 mmol, 1.11 eq) of mL of ethyl acetate. The organic extracts were combined, triethylamine and cooled to -78° C. Next, 0.080 mL (0.475 washed with 5 mL of brine, dried over anhydrous NaSO, mmol. 0.98 eq) of trifluoromethanesulfonic anhydride was filtered, and concentrated under reduced pressure. HPLC added and the mixture stirred at -78°C. for 30 minutes, then analysis of the residue indicated two peaks of almost equal warmed to 0°C. for a period of 7 minutes before use. In a area at 4.496 and 8.262 minutes. TLC analysis (25:75 ethyl separate reaction flask, 0.0338 g (0.845 mmol. 1.7 eq) of acetate:hexane on silica, CAM stain) indicated the product NaH as a 60% by wt. dispersion in oil was suspended in 3 mL with R, 0.30 and a small amount of starting material with of DMF at 0°C. Next, 0.1288 g (0.510 mmol, 1.05 eq) ofI3-d Rf 0.15. The resulting residue was purified by silica gel dissolved in 2 mL of DMF was added to the suspension. The column chromatography to obtain 0.0564 g (0.116 mmol. resulting mixture was stirred for 20 minutes at 0°C. At the end 23.9% yield) of the desired product. Mass spec (turbo spray): of this period, the “triflate” solution was added slowly to expected m/z. 487.3 M+H, observed m/z, 487.5; expected the'alkoxide’ mixture and the reaction warmed to RT. The m/z. 509.3 M+Na), observed m/z 509.5; and expected m/z. reaction was quenched by the addition of 0.200 mL of water. 995.6 2M--Na), observed m/z 995.6. After purification, The majority of the DMF and CHCl were removed by HPLC analysis indicated a single peak at 8.155 minutes. US 2015/001 7246 A1 Jan. 15, 2015

0955. In a separate experiment, THF solvent was used was obtained. Mass spec (turbo spray): expected m/z 397.2 instead of DMF. A lower yield of 13% was obtained. M+H, observed m/z 397.5; expected m/z. 419.2 M+Na), 0956 Compound I18-a: observed m/z. 419.5; and expected m/z 815.4 2M--Na. 0957 0.050 g (0.12 mmol. 1 eq) of I17-a was dissolved in observed m/z 815.8. Also present were signals consistent 2 mL methanol and 2 mL THF. The solution was purged with with a Substance on which both benzyl protecting groups had nitrogen for 15 minutes to remove dissolved gasses, followed been removed: expected m/z 3.07.2 M+H, observed m/z. by the addition of 0.105 g of Pd/C 10% wt./wt. The system 307.4; expected m/z. 419.2 M+H, observed m/z. 419.5. was sealed and purged with nitrogen gas. Next, 0.100 mL HPLC indicated a main product peak at 5.627 minutes. This (1.06 mmol, 8.8 eq) of 1,4-cyclohexadiene was added at RT. material was used in the next step without further purification. The mixture was stirred and the progress of the reaction 0958 Compound I21-a: monitored by TLC. After 1 hour, an additional 0.100 mL (1.06 0959. The tetramer is prepared following the same proto mmol, 8.8 eq) of 1,4-cyclohexadiene was added. After 2 hrs, col as described for compound I17-a from dimer I 17-8. The an additional 0.100 mL (1.06 mmol, 8.8 eq) of 1,4-cyclo compound is purified by silica gel chromatography. The iden hexadiene was added. After 3 hours, an additional 0.400 mL tity of the product is checked by mass spec and the structure (4.24 mmol. 35.3 eq.) of 1,4-cyclohexadiene was added and confirmed by 'H-NMR. The purity is checked by TLC or the reaction stirred overnight. TLC indicated consumption of HPLC. the starting material and the presence of product with R=0.30 (40:60 ethyl acetate:hexane on silica). The Pd/C was removed Example 19 by filtration of the reaction mixture through a Celite 545 pad, followed by the addition of 1 mL of MeOH and 0.75 mL of Synthesis of a Single MW SA Macromolecule (>20 water to facilitate the transfer of the product through the KDa) Crosslinking Reagent Using TBDMS Celite. The THF and MeOH were removed by evaporation Protection under reduced pressure and the water removed by lyophiliza tion. A total of 0.0365 g (0.092 mmol. 89% yield) of material 0960|

Scheme 25

CFSOOSOCF -e- Pyridine ÖBn 5Bn Bn = CH5CH2CO I3-e I3-e NaH, THF OBn OBn OBn OBn OBn OBn OBn OBn O I2, MeOH O TBDMSO Y OH -- TBDMSO Y OTBDMS OBn OBn OBn OBn OBn OBn OBn OBn I18-4a I17-4a

CFSOOSOCF Pyridine

O OBn OBn OBn OBn A OBn OBn OBn OBn O S-CF3 - - O TBDMSO O \, NaH, TBDMSO OTBDMS OBn OBn OBn OBn THF

OB gBn OB gBn Repeat 5 OB gBn OB gBn O times O TBDMSO 1, OH - TBDMSO OH OBn OBn OBn OBn OBn OBn OBn OBn I3-eb I18-4b 1) Boc-N-OHDBU 2) Pd/C/4% formic acid 3) I2/MeOH 4) TFA/H2O US 2015/001 7246 A1 Jan. 15, 2015 82

-continued

MW: 21045 Da. p3 4a-128

0961 Compound I3-e: 0967 Fmoc Protection of D-Glucosamic Acid: 0962 Compound I3-e is prepared by benzylation of I1-a 0968 Water (5 mL) was added to a stirred suspension of prepared following the same protocol as described for com D-glucosamic acid (101.3 mg 0.519 mmol), Fmoc-OSu (192 pound I5-d from I8-d (example 5). The compound is purified mg, 1.1 equiv.), and potassium bicarbonate (144 mg, 2 equiv.) by Silica gel chromatography. The identity of the product is in DMF (5 mL). Heat was generated. After sonication and the checked by mass spec and the structure confirmed by addition of more DMF (1 mL), the mixture briefly turned H-NMR. The purity is checked by TLC or HPLC. clear before white turbidity formed. The reaction was stirred at RT for 4 hours. The mixture was adjusted to pH 8 by the 0963 Compound p3 4a-128: addition of 0.5 NHCl, and then concentrated in vacuo. The 0964 Dimer I17-4a is prepared following a similar proto residue was taken up in water (~40 mL) and extracted three col as I17-a. I3-e reacted first with trifluoromethanesulfonic times with 15 mL of ethyl acetate. The aqueous layer was anhydride to form a triflate. Other good leaving groups, such adjusted to pH 2.5 by the addition of 0.5 NHCl and extracted as mesylate, can also be used here. Next, the triflate reacted three times with 30 mL of ethyl acetate. The combined with another I3-e in the presence of a base, such as NaH, organic layers were dried over Sodium sulfate, filtered, and lithium bis(trimethylsilyl)amide, or potassium tert-butoxide, concentrated to obtain the desired product (105 mg, 48%). in the presence of THF. In some cases, DMF may be used. HPLC (TFA05 method, detected by UV absorbance at 220 After the coupling, the product is purified by silica gel chro nm) retention time=5.06 min. The UV spectrum was charac matography and analyzed by HPLC and mass spec. One of teristic of the Fmoc moiety. The ESI-MS m/z was consistent the TBDMS groups of the dimer I17-4 is deprotected using with M-OH",400; M+H", 418; M+Na", 440. 1% I in MeOH following the same protocol as I3-a. The 0969. The free OH groups of the above mentioned Fmoc resulting dimer is activated with trifluoromethanesulfonic protected Sugar alcohol amino acids are protected as an anhydride and reacted with another dimer in the presence of acetate ester or tButyl ester. The final monomer is used to a base to form the tetramer I 17-4b. After iodine deprotection assemble SA macromolecules linked by an amide bond using of one of the TBDMS groups, the resulting material repeated a standard Fmoc peptide Solid phase synthesis strategy. the self-coupling and iodine deprotection cycle five times to obtain a mono-protected sugar alcohol with 128 SA units Conjugation Examples (I3-eb). The primary OH of I3-eb is converted to an aminooxy 0970) Examples 21-26 illustrate the general methods and following the same protocolas described in I4-db in example protocols for conjugating or modifying other molecules using 11. The final product is first debenzylated, then iodine is used Sugar alcohol crosslinkers. The advantages of Sugar alcohol to remove the TBDMS group, and finally TFA removed the crosslinkers over other crosslinkers are obvious in these Boc following previously described procedures in this patent. examples because Sugar alcohol crosslinkers are so hydro The final result is a aminooxy derivatized over a 20 KDa SA philic that little or no organic solvent is needed for the reac compound ready for conjugation through the aminooxy. tion. Particularly in the cases of antibody-drug conjugate, the sugar alcohol link will greatly reduce the hydrophobicity of Example 20 the drug and make it easy to react it with an antibody in the aqueous buffer after modification. One can also expect less Preparation of Other Monomers for Synthesizing aggregation and more stable antibody-drug conjugate. Higher MW SAS Linked by an Amide Bond Example 21 0965 Fmoc Protection of D-Glucamine: 0966 D-glucamine (1.02g, 5.52 mmol), Fmoc-OSu (1.99 Synthesis of an Antibody-Drug Conjugate g, 1.05 equiv.), and potassium carbonate (0.78 g, 1 equiv.) 0971 The standard method for conjugating an antibody to were suspended in DMF (40 mL). Water (40 mL) was added a toxin is through a maleimide-NHS crosslinker (according to in Small portions, generating heat. The reaction mixture most published methods in the antibody-drug conjugate briefly turned clear, and then a precipitate started to form. area). The disulfide bonds in the hinge region of the antibody After stirring overnight, the reaction mixture was poured into can be selectively reduced to generate a half antibody con 750 mL of water. Precipitate was collected by suction filtra taining Sulfhydryl groups, which then react with a maleimide tion and dried by lyophilization to obtain a yellow solid (1.75 modified toxin. Antibodies can also be thiolated at a surface g), of which approximately 40% was the desired material amine to generate a sulfhydryl group, which can then be according to the HPLC peak area. HPLC (TFAO5 method, reacted with a maleimide-modified toxin. The second most detected by UV absorbance at 220 nm) retention time=5.10 popular method involves NHS ester coupling. For example, if min. The UV spectrum was characteristic of the Fmoc moiety. the toxin has a free acid, it can be coupled to the Surface amine ESI-MS of the fraction collected from HPLC: m/z consistent of the antibody through the NHS ester activation method with M+H, 404: M+Na, 426. (simplified process). US 2015/001 7246 A1 Jan. 15, 2015

0972 CD33 Antibody-Doxorubicin: terminal modified amine oligo is dissolved in sodium phos 0973 Commercially available CD33 antibody and doxo phate buffer (0.1 M. pH 8.0-9.0) at 10-20 mM concentration. rubicin are used to demonstrate the feasibility of synthesizing An SA macromolecule bearing a single NHS ester is added antibody-drug conjugates using a mannitol-based crosslink directly into the oligo solution with a final concentration of ing reagent. In methoda, the antibody is reduced at the hinge SA macromolecule in the range of 1-2 mM. The vial is placed region to yield two half-antibody molecules, which react with in a shaker at 37° C. for a few hours. The progress of the maleimide-activated doxorubicin through a maleimide man reaction can be checked by size-exclusion HPLC, anion nitol aminooxy crosslinking reagent. In method b, doxorubi exchange, or reversed-phase C18 chromatography. Depend cin is reacted with carboxylic acid mannitol aminooxy, and ing on the size of the oligo, the reaction mixture can be then reacted with the surface amines of the antibody through desalted using gel filtration (SuperdexTM 200) or ultrafiltra EDC. After conjugation, the reaction mixtures are dialyzed or tion to remove the unreacted oligo. If the MWs of the oligo desalted to remove the unconjugated drug. Unconjugated and SA are very close, a 1:1 ratio of the oligo and SA should (free) antibody is removed through preparative hydrophobic be mixed together and the product purified by anion exchange interaction chromatography (HIC) purification. The number chromatography. A 5' or 3' thiol modified oligo is also conju of drug molecules loaded onto the antibody (antibody-drug gated to an SA macromolecule (>20 KDa) bearing a single ratio) is determined by HPLC analysis using a HIC column. maleimide group at the termini following the standard proto The MW of the antibody-drug is analyzed by matrix-assisted col. The identity of the product is checked by MALDI-MS. laser desorption/ionization (MALDI) or electrospray mass Gel electrophoresis is used to analyze the reaction. Please spectrometry. The exact position of the labeling is determined note that sugar alcohols have no UV absorbance and may by trypsindigestion of the antibody and analyzed by LC/MS require a special detector, such as a pulsed electrochemical MS/MS. The percentage of aggregation is determined by gel detector (PAD), refractive index detector, or light scattering filtration chromatography. detector. Other methods are spotting the sample in TLC and charring by CAM or PMA reagents. Example 22 Synthesis of Single Pure SA Macromolecules and Example 25 Protein Conjugates Synthesis of a Single Pure SA Macromolecule and 0974. This example illustrates tetra-mannitol labeling of Peptide Conjugates human granulocyte-colony stimulating factor (rhG-CSF). Recombinant rhG-CSF is prepared according to the literature 0977. An SA macromolecule (>20 KDa) can easily be (Souza, L. M. et al. Science 1986, 232, 61-65). A typical attached to a thiol peptide using the following procedure. An protocol for preparing single labeled rhG-CSF thorough the SA macromolecule bearing a single maleimide group is terminal amine is illustrated. A solution of rhG-CSF (5 mixed with 5-10 equivalents of thiol peptide in sodium phos mg/mL) in sodium phosphate buffer (pH 6.5) at 4°C. is added phate buffer (0.1 M. pH 6.0) and mixed at RT. The progress of to a vial containing mannitol carboxymethyl-N-hydroxysuc the reaction is checked by size-exclusion HPLC, anion cinimidyester (18 equiv.). After the SA molecule is dissolved, exchange, cation exchange, or reversed-phase C18 chroma the reaction is stirred at 22°C. for 1 hr. Hydroxylamine (2 M, tography. Depending on the size of the oligo, the reaction pH 7.3) is added to the reaction mixture to cleave any unstable mixture is desalted using gel filtration (SuperdexTM 200) or SA labeling sites on rhG-CSF. After 1 hr, the reaction mixture ultrafiltration to remove the unreacted peptide. If the MWs of is diluted with 1 mM HCl and the pHadjusted to 3.5 with 1M the oligo and SA are very close, a 1:1 ratio of the peptide and HCl, rhG-CSF-SA is purified by cation exchange chromatog SA should be mixed together and the product purified by raphy. The site of SA labeling is determined by endoprotein anion or cation exchange chromatography. Other peptides, ase peptide mapping. The MW of the intact rhG-CSF-SA is Such as keto peptide, are also synthesized and react with a SA determined by MALDI. macromolecules (>20 KDa) bearing an aminooxy group under acidic conditions (usually acetic acid in MeOH) to Example 23 obtain a SA-peptide conjugate. Synthesis of Highly Loaded Therapeutic Agents on SA Macromolecules Example 26 0975 Doxorubicin has a ketone group that easily reacts with aminooxy-containing SA macromolecules. An SA mac Synthesis of a HRP-Oligo Using a Sugar Alcohol romolecule containing multiple copies of aminooxy is Heterobifunctional Crosslinker reacted with doxorubicin to produce a highly loaded drug. Tetra-mannitol containing aminooxy in place of secondary 0978. A 5' or 3' modified amine oligo is first reacted with a Sugar alcohol with an NHS-ester on one end and 2-py OH groups reacts with doxorubicin, producing a highly ridyldithiol on the other end (MW-500 Da) in sodium phos loaded therapeutic agent. phate buffer (pH>8.0). The reaction is typically performed at Example 24 37°C. for 4hr. The reacted oligo is desalted on a SephedexTM G25 column and freeze-dried. The dried oligo is then dis Synthesis of a Single Pure SA Macromolecule and solved in sodium phosphate buffer (pH>8.0) and reduced Oligo Conjugates with DTT at 37° C. for 30 minutes. The reduced oligo is desalted and added directly into an HRP-bearing maleimide 0976 An SA macromolecule (>20 KDa) can easily be group. Maleimide-HRP is prepared by reacting HRP with an attached to an oligo using the following procedure. A 5' or 3' NHS-maleimide sugar alcohol crosslinker (MW<500 Da) or US 2015/001 7246 A1 Jan. 15, 2015 84 any other NHS-maleimide crosslinker. The oligo and HRP 0982 Specifically, the method consists of: conjugation reaction is allowed to proceed at RT and its (i) providing the first mono-SA unit having the general struc progress is monitored by gel filtration (or anion exchange) tural formula: chromatography. The HRP-oligo conjugate is purified by gel filtration or anion exchange chromatography to obtain a single oligo-labeled HRP-containing Sugar alcohol spacer. OB

0979 The following list additional methods and examples DMTC- sus-onpi for synthesizing various SA units, single MW SA polymers, and crosslinking reagents. 0983. Wherein 0980 Example 19 described a specific method for synthe 0984 n is an integer from 1 to about 8: 0985) Bn represents a benzyl ether and DMTC is dim sizing higher MW linear SA molecules using TBDMS pro ethylthiocarbamate. tection strategy. However, in general it was found out the (ii) providing the second mono-SA unit having the general method is not practical or efficient to obtain higher order SA structural formula: macromolecules. For example, the method uses trifluo romethanesulfonic acid anhydride to activate the free OH group. Trifluoromethanesulfonic acid anhydride is a very reactive reagent and the reaction conditions are very difficult to control. If not careful, side products are generated during T1 iii. the activation, despite using a dry icefisopropanol bath (-80° C.). The method also uses a strong base, NaH, to deprotonate 0986 Wherein the hydroxyl group. Sometimes the TBDMS protecting group 0987 m is an integer from 2 to about 8: comes off during the coupling reaction, leaving unwanted 0988) Bn represents a benzyl ether and Trt represents a side products. trityl, or substituted trityl that having one or several 0981 To improve the overall yield so that a higher order functional groups such as methoxy on the aromatic ring. SA macromolecule can be generated efficiently, a specific (iii) substituting the primary OH group of the first mono-SA unit with a good leaving group Such as mesylate ester. method for assembling the O-linked SA macromolecules was (iv) deprotonate the free OH group of the second mono-SA tested (Scheme 26, for general method please see FIG. 6). The unit in the presence of a base, such as NaH, LiHMDS, or method uses three sets of orthogonal protecting groups (ben potassium tert-butoxide Zyl ether, trityl or substituted trity1, and dimethylthiocarbam (V) combining the first mono-SA unit with the second mono ate) for the OH groups in the Sugar alcohol monomers. Two SA unit under conditions that permit the condensation of sets of the Sugar alcohol precursor units containing one of the these two units to form a dimer. primary OH groups protected by either a trity1 or substituted and (vi) deprotecting one of the primary OH groups of the trityl, or dimethylthiocarbamate (DMTC) group and the sec dimer to provide a di-SA building block (two SA units) that ondary OH groups are protected as benzyl ether, are synthe can be utilized to build a higher MW SA molecule. Higher sized. Iterate coupling of the Sugar alcohol precursor units orders of SA building blocks (greater number of SA units) can followed by selective deprotection of the trity1 or dimethylth be built following repetitive steps (i) to (vi) using r-SA build iocarbamate group of the resulting product efficiently gener ing blocks, where r represents the number of SA units in one ate higher order O-linked Sugar alcohol marcomolecules. molecule.

Scheme 26:

OB

i) Mic-out-oilpi 1st mono-SA unti n Activation

OB DMTC- sub pi

w) Condensation US 2015/001 7246 A1 Jan. 15, 2015 85

-continued OB OB

DMTC- N--N--pi iii. n Trt

vi) Deprotection

OB OB OB OB

n DMTC------pi iii. "N-----pi iii. Trt 1st di-SA unit 2nd di-SA unit |

Iterate steps i to vi

OH OH OH OH Substitue. One OH DMTC- O O O-Trt -- -- HO O Y pi iii. p pi iii. p |als deprotected Nsubstitute two OH groups

OH OH OH OH

HO O OH X O Y pi iii. p pi iii. p

Protecting groups: US 2015/001 7246 A1 Jan. 15, 2015

0989 Scheme 27 describes the synthetic routes to obtain the first and second mono-SA unit for the coupling reaction in Scheme 26.

Scheme 27: OH OH Protection HO OH -as O O N--pi Protection Trt 1. N--pi n Trt

OB protection

mic-sub-orpi a- les-builtpi T1 --lpi 1st SA unti 2nd SA unit

0990 Higher MW linear SA molecules can be built by Example 27 stepwise or convergent, or stepwise plus convergent methods depending on the heterogeneity of the secondary alcohol Synthesis of the First and Second Mono-SA Units groups in the SA molecules. For example, tetra-SA building for the Condensation Reactions Using Mannitol as blocks containing different numbers and different kinds (dif an Example ferent stereochemistry) of secondary OH groups on each SA unit can be built by coupling four different mono-SA units in 0993 a stepwise manner. Tetra-SA building blocks with different numbers and different kinds of secondary OH groups on each Scheme 28: Synthetic route to the Trt mono-SA unit using SA unit can also be built by coupling two units stepwise first, mannitol as an example. and then converging two of the di-SA building blocks OH OH together. Tetra-SA units with the same number and same s TrC1, Pyridine kinds of secondary OH groups on each SA unit can be built by OH --> coupling two di-SA building blocks. In most cases, the low

MWSA building blocks are built stepwise and the high MW SAS are assembled by convergence. 0991 Functional groups X and Y can be assembled into the termini group by substituting the primary OH with desired crosslinking groups at the termini according to the methods described in this invention. 0992. The following examples 27-39 describe specific methods of synthesizing varieties of low MW SA building units and using these SA units to assemble a single MWSA polymer. Three sets of orthogonal protecting groups (benzyl ether, trityl or substituted trityl, and dimethylthiocarbamate) I2-Bn-T for the OH groups have been described in details here. Other SOH DCM/MeOH, OH protecting groups can also be used for assembling SA RT molecules based on these methods, however the deprotection conditions for three protection groups (PG1, PG2, and PG3 in FIG. 6) have to be orthogonal to each other. An extensive description of protecting groups of typical art can be found in: Theodora W. Green and Peter G. M. Wuts, Protective Groups in Organic Synthesis, 2" ed., Wiley-Interscience, New York, 1991. Examples of OH protecting groups include, but are not limited to, alkyl, aryl, benzoyl, acetyl, benzyl, alkyl silyl, I4-Bn-T tetrahydropyranyl, tetrahydrofuranyl, and tetrahydrothio 2nd mono-SA unit furanyl. US 2015/001 7246 A1 Jan. 15, 2015 87

-continued (yield: 109%). The product was used directly for the next reaction without further purification. Mass spec analysis (MALDI-TOF): exact mass: 1026.49, obtained: 1050.85 M+Na". 1-O-trityl-6-OH-2,3,4,5-tetra-O-benzyl-D-mannitol (I4-Bn-T) (2nd SA Unit) 0996 277 mL of DCM was added to a 1 L round-bottom flask containing 18 g (17.52 mmol) of 1,6-di-O-trityl-2,3,4, 5-tetra-O-benzyl-D-mannitol. The reaction mixture was Scheme 29: Synthetic route to the DMTC mono-SA unit using stirred for a few minutes until a clear solution was obtained, mannitol as an example. then 146 mL of MeOH was added, followed by 1.67 g (10.6 mmol) of benzene sulfonic acid. The reaction mixture was QN. N-4?y 2M stirred at RT overnight and concentrated under reduced pres OB OB Dimethylamine Sure and purified by silica gel chromatography (EtOAC/hex S N in THF OH Her anes) to obtain 2.73 g of mono-trityl deprotected mannitol HO DMAPDCMRT (1-O-trityl-2,3,4,5-tetra-O-benzyl-D-mannitol) (yield: OBn OBn 20%). Mass spec analysis (electrospray): exact mass 784.38. I8-Bn obtained mass 806.8 M+Na". H-NMR (500MHz, CDC1): 8 7.48-7.53 (m, 6H, Trt-H), 7.20-7.40 (m, 26H, aromatic H), 7.07 (dd. 3H, Trt-H), 4.81 (d. 1H, CH, CH ), 4.70 (d. 1H, -CHs CH ), 4.60(dd. 3H, —CH-CH ), 4.50 (d. 1H, -CHS CH ), 4.43 (dd. 2H. —CHS CH ), 4.12 (m. 1H, Trt-O-CH ), 4.06 (m. 1H, Trt-O-CH ), OB 3.90 (m, 2H, -CH-OH), 3.80 (m. 1H, -CH ), 3.71 (m, 1st mono-SA unit 1H, -CH ), 3.65 (m. 1H, -CH ), 3.40 (dd. 1H, - C I4-Bn-D H ); 'C-DEPT135 (500 MHz, CDC1,) 128.83, 128.50, 128.37, 128.28, 128.13, 127.80, 127.74, 127.70, 127.69, 127. 50, 127.48, 127.45, 127.34, 126.97, 79.62 ( CH ), 78.91 1,6-di-O-trityl-D-mannitol (Il-T) ( CH ), 78.68 (-CH ), 78.40 (-CH-), 74.33 0994 Five grams (27.44 mmol) of D-mannitol, 60 mL of (—CHS CH2), 73.82 (-CHS CH), 72.01 (—CHs pyridine, and 134 mL of dichloromethane (DCM) was added CH ), 71.33 (-CH, CH4), 62.51 (Trt-O-CH), 60.49 to a 250 mL 3-neck round-shaped flask, followed by 16.9 g of (CH-OH). triphenylmethyl chloride. The reaction mixture was refluxed 1,6-OH-2,3,4,5-tetra-O-benzyl-D-mannitol (I8-Bn) for 12-24 hrs until all of the starting material disappeared. The reaction mixture was transferred to a 500 mL reparatory 0997. A total of 2.09 g of di-trityl deprotected mannitol funnel and washed once with 100 mL of water. The water (2,3,4,5-tetra-O-benzyl-D-mannitol) was obtained as a side layer was back extracted three times with DCM. The DCM product from the deprotection reaction of 18 g of 1,6-di-O- layers were combined and washed three times with brine. The trityl-2,3,4,5-tetra-O-benzyl-D-mannitol (yield: 42%). Mass DCM layers were dried over anhydrous NaSO filtered, and spec analysis (electrospray): exact mass 542.27, obtained concentrated under reduced pressure. The resulting residue mass 564.7 M+Na". H-NMR (500 MHz, CDC1): 8 7.28 was dissolved in a minimum amount of DCM and subjected 7.4 (m, 20H. —CH ), 4.81 (d. 2H. —CHS CH ), to silica gel column chromatographic purification (EtOAC/ 4.70 (d. 2H. —CHs CH ), 4.60 (d. 2H. —CHs C hexanes) to obtain 16.4 g of the desired product (89.6% H ), 4.45 (d. 2H. —CHS CH ), 3.96 (m, 3H, —C yield). Mass spec analysis (electrospray): exact mass 666.3, H ), 3.96 (m. 1H, -CH ), 3.86 (m. 1H, -CH ), 3.84 obtained mass 688.8 M+Na". (m, 1H, -CH ), 3.68-3.74 (m, 2H, -CH ); 'C- DEPT135 (500 MHz, CDC1,) 128.56, 128.41, 127.97, 127. 1,6-di-O-trityl-2,3,4,5-tetra-O-benzyl-D-mannitol 92,127.77, 127.74, 79.71 ( CH ), 78.87 ( CH ), 74.50 (I2-Bn-T) (—CHS CH2—), 71.53 (-CHS CH2—), 60.45 (— 0995) 10.64 g (16 mmol) of 1,6-di-O-trityl-D-mannitol CH, OH). and 3.07 g of NaH (60% oil dispersion) were added into a 250 1-OH-6-O-DMTC-2,3,4,5-tetra-O-benzyl-D-manni mL round-bottom flask. The flask was purged with N and cooled in a dry icefisopropanol bath, and then 133 mL of tol (I4-Bn-D) anhydrous DMF was added. After stirring for 5 min, 8.55 mL 0998 0.48 g (2.68 mmol) of 1,1'-thiocarbonyldiimidazole (72 mmol) of benzyl bromide was added slowly to the reac and 31 mg (0.24 mmol) of dimethylaminopyridine (DMAP) tion mixture. The dry ice/iPrOH bath was removed and the was added to a 50 mL round-bottom flask containing 1.32 g reaction mixture stirred at RT for 3.5 hr and then concentrated (2.44 mmol) of 1,6-OH-2,3,4,5-tetra-O-benzyl-D-mannitol. under reduced pressure. 400 mL of EtOAc and 300 mL of After flushing the flask with N. 13 mL of anhydrous DCM deionized water were added to the residue. After separation, was added. The reaction was stirred at RT overnight and then the water layer was back extracted twice with EtOAc. The the solvent removed under reduced pressure. 10.52 mL of 2M combined EtOAc layers were washed three times with brine, dimethylamine in THF was added to the residue. After over dried over anhydrous NaSO, filtered, and concentrated night stirring at RT under N, the solvent was removed in under reduced pressure to obtain 18 g of desired product vacuo and the residue chromatographed over silica gel