Patent Application
20140227793
This disclosure is directed to glycan reagents and the use of the glycan reagents to determine the structure of target glycans having a reducing terminus.
Glycans are one of the four families of structurally related macromolecules that comprise living organisms, along with nucleic acids, proteins, and lipids. However, unlike DNA, RNA, and proteins, which possess predominantly linear structures comprising a limited number of subunits with defined stereochemistry, glycans may exhibit complicated branched structures with a large number of subunits having both structural and stereochemical diversity. As a result, the field of glycomics is much less developed than genomics and proteomics. Nevertheless, genetic and biochemical studies over the past several decades have established the importance of glycans in many fields, including various aspects of health, such as immunity response, inflammation signaling, and disease prevention, as well as green energy production and materials fabrication. Therefore, understanding the structure and function of glycans will complement and strengthen other areas of research.
Mass spectrometry (MS), noted for its minimal sample consumption, high sensitivity, and short acquisition time, has been widely employed for structural characterization of glycans. Moreover, mass spectrometers allowing for tandem mass spectrometry (MSn) have been used extensively as indispensable tools for glycan structural analysis. Ionization efficiency is especially important for the MSn experiments to achieve good sensitivity and wide dynamic ranges. However, due to lack of strongly basic sites for protonation, glycans are traditionally difficult to ionize. (Edge et al. Nature 1992, 358, 693-694, the entire contents of which are herein incorporated by reference.)
Using suitably ionized glycans, many techniques exist for effecting dissociation processes that provide structural information. Low-energy collision-induced dissociation (CID) typically generates glycosidic bond cleavage when applied to glycans. Infrared multiphoton dissociation (IRMPD), another slow-heating fragmentation method, gives results similar to low-energy CID. (Harvey, D. J. J. Am. Soc. Mass Spectrom. 2001, 12, 926-937; Adamson, J. T.; Hakansson, K. Anal. Chem. 2007, 79, 2901-2910; and Xie, Y. M.; Lebrilla, C. B. Anal. Chem. 2003, 75, 1590-1598, the entire contents of all of which are herein incorporated by reference.) High-energy CID and vacuum ultraviolet multiphoton dissociation are unavailable on many modern instruments, even though they can generate more cross-ring cleavages than low-energy CID and IRMPD. More recently, techniques including electron capture dissociation (ECD), electron detachment dissociation (EDD), and electron transfer dissociation (ETD) have been demonstrated to provide extensive and complementary information about glycan structure. (Zhang et al., J. Proteome Res. 2009, 8, 734-742; Budnik et al., Anal. Chem. 2003, 75, 5994-6001. Zhao et al., J. Am. Soc. Mass Spectrom. 2008, 19, 138-150; Wolff et al., Anal. Chem. 2010, 82, 3460-3466; and Han et al., C. J. Am. Soc. Mass Spectrom. 2011, 22, 997-1013, the entire contents of all of which are herein incorporated by reference.)
In contrast to the often unpredictable dissociation pathways and yields associated with the above techniques, natural enzymes excel in depolymerizing glycans into their components, often in a systematic and predictable manner, by taking advantage of acid-base catalysis to achieve selective cleavage of the glycosidic bond. In fact, enzymatic structural analysis of glycans employing a set of highly specific exoglycosidases, sequentially or in a matrix array, has proven to be a powerful analytical tool for the determination of sequence, linkage type, and anomeric configuration. However, this method requires highly pure samples, fully completed hydrolysis, and lengthy enzymatic incubation periods. In addition, certain glycosidic bonds, particularly those between two glucose residues, are highly resistant to enzymatic degradation due to their high stability. (Edge et al., supra; and Wolfenden et al., J. Am. Chem. Soc. 2008, 130, 7548-7549, the entire contents of both of which are herein incorporated by reference.)
The efficiency of these natural enzymes provides an impetus to the development of biomimetic reagents that, when combined with MS, attempt in part to replicate their chemistry while eliminating the shortcomings of a purely enzymatic approach to glycan sequencing.
In embodiments of the present invention, a glycan reagent for depolymerization of a glycan having a reducing end is represented the formula MCX, wherein M is a glycan coupling group selected from oxylamines and hydrazides; C is a fixed charge group or a basic group having a proton affinity of 210 kcal/mol; and X is hydrogen or a free radical initiator.
In some embodiments, the glycan reagent is represented by one of the following formulas:
In the above formulas, X is hydrogen or a free radical initiator, R is hydrogen, an alkyl, or an alkoxy, R1 and R2 are each independently an alkyl, and m and n are each independently an integer.
In some embodiments, a method of depolymerizing a glycan having a reducing terminus includes conjugating the glycan reagent of claim 1 to the reducing terminus of the glycan to form a derivatized glycan, ionizing the derivatized glycan to form an ionized derivatized glycan, and dissociating the ionized derivatized glycan to form fragment glycan ions.
In some embodiments, a method of depolymerizing a glycan having a reducing terminus includes conjugating a glycan reagent to the reducing terminus of the glycan to form a derivatized glycan, isolating the derivatized glycan to form an isolated derivatized glycan, ionizing the isolated derivatized glycan to form an ionized derivatized glycan, and dissociating the ionized derivatized glycan to form fragment glycan ions.
In some embodiments, a method of identifying subunit connectivity of a glycan having a reducing terminus includes conjugating a glycan reagent represented by one of structures (a) through (j) to the reducing terminus of the glycan to form a derivatized glycan, ionizing the derivatized glycan to form an ionized derivatized glycan, dissociating the ionized derivatized glycan to form fragment glycan ions, and analyzing the fragment glycan ions to determine the subunit connectivity of the glycan:
In the above structures, X is hydrogen, R is hydrogen, R1 and R2 are each independently an alkyl.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Aspects of the present invention are directed to a glycan reagent that utilizes acid-base chemistry and/or free radical chemistry for depolymerization and sequencing of a glycan having a reducing terminus as depicted in
As used herein, “depolymerization” and “fragmentation” are used interchangeably and with respect to a glycan, refer to the conversion of the glycan into its subunits. Depolymerization using a glycan reagent as disclosed herein is ordered, and therefore, the depolymerization of the glycan allows for determination of the sequence of the glycan subunits. As such, sequencing of a glycan is the observation of the depolymerization. Additionally, “sequencing of a glycan,” “glycan sequencing,” and “glycan determination” are used interchangeably to refer to the elucidation of a glycan structure resulting from depolymerization (or fragmentation) of the glycan.
As used herein, all product ions are classified according to the Domon and Costello nomenclature (Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409, the entire contents of which are herein incorporated by reference). The Domon and Costello nomenclature for product ions is shown schematically in
In some embodiments of the present invention, a glycan reagent for ordered depolymerization of a glycan having a reducing terminus includes a compound represented by the formula M-C—X, in which M is a moiety capable of coupling to the reducing end of the glycan, C is a moiety having a fixed charge, and X is either hydrogen (H) or a free radical source (e.g., a free radical initiator). In some embodiments, the glycan coupling group M may be selected from oxylamine groups (—ONH2) and hydrazides (—NHNH2). In some embodiments, C is a fixed charged group or a basic group having a proton affinity of at least 210 kcal/mol. When C is a fixed charge group, the charge remains on the reagent when coupled to the reducing end of the glycan. For example, the C moiety includes: cationic species with and without a labile proton; anionic species that have conjugate bases of neutral acids (e.g., proton acceptors) and anionic species that have a fixed negative charge. In some embodiments the C moiety of the MCX reagent has a proton affinity of at least 210 kcal/mol. For example, the free radical source X includes any suitable free radical initiator that is chemically coupled to C. Non-limiting examples of free radical initiators include peroxides, nitrites, alkoxyamines, and azo compounds. Specific examples of free radical initiators include TEMPO and 4,4-azobis(4-cyanopentanoic acid) (Vazo 68, Dupont).
In some embodiments of the present invention, the glycan reagent of the present invention utilizes acid-base depolymerization, free radical activated depolymerization, or a combination of both acid-base and free radical chemistries. An example of an acid-base catalyzed glycan depolymerization compound is referred to as Proton Reagent for Acid-catalyzed Glycan Sequencing (PRAGS). An example of a free radical based glycan depolymerization compound is referred to as Free Radical Activated Glycan Sequencing I (FRAGS I), and an example of an acid-base and free radical glycan depolymerization compound is referred to as Free Radical Activated Glycan Sequencing II (FRAGS II), as shown in
Similar to enzymatic glycosidic bond cleavage, PRAGS employs acid-base chemistry to effect selective C1-O glycosidic bond cleavage (Y ions), with the charge retained on the reducing terminus. With both a labile proton and radical precursor, collisional activation of FRAGS II-derivatized glycans yields abundant cross-ring as well as glycosidic bond cleavages, resulting from both free radical and acid-base chemistry, with charge retention at the reducing terminus. Additionally FRAGS I-derivatized glycans eliminate the acid-base reaction, using only free-radical chemistry. The difference between FRAGS I and FRAGS II fragmentation is evidenced by the disappearance of the Y* ion and Y+Y ion, thereby decreasing the complexity of the spectra while keeping all the essential fragmentation patterns for the structure determination of glycans. All major fragments are products of Y- and Z-type glycosidic bond cleavages, and 1,5X cross-ring cleavages. Additionally, the specific fragmentation pattern (Z+Z+H), resulting from the free radical chemistry of the FRAGS I, is observed only at the branch site, providing the information to confirm presence of the branch structure.
Example compounds for each of PRAGS, FRAGS I, and FRAGS II reagents are shown below.
An example of a Proton Reagent for Acid-catalyzed Glycan Sequencing (PRAGS) reagent is shown above, in which the glycan coupling group M is O—NH2 as shown in the dotted box. A PRAGS reagent does not include a free radical source group, and as such, X is hydrogen. In the above PRAGS example, the fixed charge group C is methylpyridine.
An example of a FRAGS I reagent is shown above in which the glycan coupling group M is O—NH2 as shown in the dotted box. In this example, the fixed charge group C is a N-methyl-methylpyridinium ion group. The methylation of the pyridine N (nitrogen) atom eliminates the acid-base reaction found in PRAGS and FRAGS II compounds. In the FRAGS I example above, X is (2,2,6,6-Tetramethylpiperidin-1-yl)oxy (TEMPO), shown above in brackets. The bond that is cleaved to produce a free radical between N-methyl-methylpyridinium and TEMPO is shown in bold.
An example of a FRAGS II reagent is shown above in which the glycan coupling group M is O—NH2 as shown in the dotted box. In this example, the fixed charge group C is a methylpyridine and X is TEMPO, shown above in brackets. The bond that is cleaved to produce a free radical between N-methylpyridinium and TEMPO is shown in bold.
In some embodiments of the present invention, the glycan reagent may be a PRAGS, FRAGS I or FRAGS II compound. Non-limiting examples of PRAGS, FRAGS I and FRAGS II compounds are represented by the following structures:
For a PRAGS reagent, for each of structures (a) through (h) above, X is hydrogen and R is hydrogen. For a FRAGS I reagent, for each of structures (a) through (i) above, X is a free radical initiator (e.g., TEMPO-CH2) and R is an alkyl group or an alkoxy group. For a FRAGS II reagent, for each of structures (a) through (i) above, X is a free radical initiator (e.g., TEMPO-CH2) and R is hydrogen. For a FRAGS I reagent, for each of structures (a) through (i) above, X is a free radical initiator (e.g., TEMPO-CH2) and R is alkyl or alkoxy. For PRAGS, FRAGS I, and FRAGS II reagents represented by structure (i), n is any integer so long as the radical group interacts with the glycan and does not react with the reagent itself. In some embodiments n is an integer from 1 to 3.
In some embodiments of the present invention, the glycan reagent may be a PRAGS, FRAGS I or FRAGS II compound represented by one of the following structures:
For a PRAGS reagent, for each of structures (i) and (j) above, X is hydrogen, R is hydrogen, and R1 and R2 are each independently an alkyl. For a FRAGS I reagent, for each of structures (i) and (j) above, X is a free radical initiator (e.g., TEMPO-CH2), R is alkyl or alkoxy, and R1 and R2 are each independently an alkyl. For a FRAGS II reagent, for each of structures (i) and (j) above, X is a free radical initiator (e.g., TEMPO-CH2), R hydrogen, and R1 and R2 are each independently an alkyl. For PRAGS, FRAGS I, and FRAGS II reagents represented by structures (i) or (j), both m and n are each independently any integer so long as the radical group interacts with the glycan and does not react with the reagent itself. In some embodiments m and n are each independently an integer from 1 to 3.
In some embodiments of the present invention, the glycan reagent may be a FRAGS I reagent represented by the following structure:
For a FRAGS I reagent, structure (k) and (j) above, X is a free radical initiator (e.g., TEMPO-CH2) and both m and n are independently any integer so long as the radical group interacts with the glycan and does not react with the reagent itself. In some embodiments m and n are independently are each independently an integer from 1 to 3.
According to embodiments of the present invention, glycan reagents represented by the structural formula MCX may be synthesized following methods known in the art. Certain specific and exemplary synthetic methods are disclosed herein (Examples 1 and 8), but the present invention is not limited thereto. In some embodiments of the present invention, an MCX glycan reagent (e.g., a PRAGS, FRAGS I, or FRAGS II reagent) is coupled to a glycan having a reducing terminus. Coupling of an MCX reagent to a glycan may be carried out by methods known in the art, certain examples of which are disclosed herein (Example 1), but the present invention is not limited thereto. The coupling of an MCX reagent to a glycan results in an “MCX-derivatized glycan”, (e.g., PRAGS-derivatized glycan, FRAGS I-derivatized glycan, or FRAGS II-derivatized glycan). The MCX-derivatized glycan may also be referred to as an “MCX-glycan conjugate”.
In some embodiments of the present invention, the MCX-derivatized glycan is ionized to form an ionized derivatized glycan. The ionized derivatized glycan is subsequently dissociated to form fragmented glycan product ions. For example, the MCX-derivatized glycan may be ionized by electrospray ionization (ESI), followed by collision-induced dissociation (CID), followed by analysis using mass spectrometry. In some embodiments of the present invention, a second CID step is performed. For example, a second CID step may be performed on a product ion generated from the first CID step.
In some embodiments, a method of deconstructing the glycans to identify subunit connectivity includes derivatizing a glycan with a PRAGS reagent followed by fragmentation as disclosed herein. A subunit as disclosed herein refers to a monosaccharide. Because the proton is retained in the PRAGS reagent, subsequent to the first glycosidic bond cleavage, repeat glycosidic bond cleavage reactions are possible. Subsequent bond cleavage reactions allow for a deconstruction of the glycan, thereby producing a DECON diagram. An example of a DECON diagram is shown in
In some embodiments of the present invention, a glycan composition includes an MCX reagent coupled to a support or an affinity tag. Using a support or an affinity tag, derivatized glycans may be effectively isolated for analysis. For example, in order to determine the glycan structure of a selected glycoprotein, the glycans are cleaved from the glycoprotein and then derivatized with an MCX reagent. An affinity tag coupled to the MCX reagent allows for isolation of the derivatized glycans from the protein using the corresponding moiety for the selected support or affinity tag. After isolation of the derivatized glycan and prior to fragmentation, the support or affinity tag is cleaved from the derivatized glycan. In some embodiments of the present invention, any suitable support or affinity tag may be coupled to the MCX reagent. Suitable supports and affinity tags are those which do not inhibit the coupling of the MCX reagent to the glycan and those which can be removed. Non-limiting examples of supports and affinity tags include resins, biotin, and histidine. Coupling of the support or affinity tag is performed using known methods. In some embodiments of the present invention, the MCX reagent is coupled to a resin support, an example using sepharose resin is described herein (Example 5), but the present invention is not limited thereto. Any suitable resin support may be used as would be understood by those having ordinary skill in the art. In some embodiments, the MCX reagent is coupled to an affinity tag such as biotin or histidine, as disclosed in U.S. patent application Ser. No. 13/135,543, the entire contents of which are herein incorporated by reference.
In some embodiments, glycans to be derivatized are first enzymatically removed from a glycoprotein. Enzymatic removal of glycans from glycoproteins, for example, N-glycans, is possible using PNGase F (Peptide-N-Glycosidase F) or Endo H (endonuclease H), as described in Maley et al. 1989, Anal. Biochem. 180: 195-204 PMID: 2510544, the entire contents of which are herein incorporated by reference. For O-linked glycans, a combination of O-glycosidase for core 1 and core 3 glycans in combination with exoglycosidases will allow for removal of large O-glycans as described in Koutsioulis et al., 2008, Glycobiology, 18, 799-805, PMID: 18635885, the entire contents of which are herein incorporated by reference.
The following Examples are presented for illustrative purposes only, and do not limit the scope or content of the present application.
The synthesis strategy for the FRAGS I reagent and FRAGS II reagent is summarized in Scheme 1 above. (Syntheses of reactants shown here in Scheme 1 are described in Example 8.) The FRAGS II reagent synthesis was accomplished by benzylic bromination with N-bromosuccinimide (NBS), coupling with TEMPO, reduction of the ester group, activation of the hydroxyl group, and finally hydrazinolysis of the imide group. For FRAGS I synthesis, methyl iodide (iodomethane) was added to the FRAGS II product. A similar synthesis strategy excluding TEMPO derivatization was employed to synthesize the PRAGS reagent (1), as shown in Scheme 2 below.
For glycan derivatization, 2 uL of a 20 mM solution of the final product (1 or 2) in acetonitrile (ACN) was mixed with 10 uL of a 1 mM solution of the glycan in H2O with 1% acetic acid (pH about 4.6). The reaction mixture was allowed to react at 60° C. for 5 hours. After desalting with C18 pipet tips according to the reported protocol, the resulting glycan conjugates were ionized by electrospray ionization (ESI) coupled with an ion trap mass spectrometer (
Maltoheptaose presents a linear chain of seven identical glucose subunits (
In the first step of Scheme 3, the protonated pyridinium cation functions as a general acid catalyst by protonating the glycosidic oxygen. In the second step, the glycosidic bond is cleaved via the participation of the lone pair of electrons on the endocyclic oxygen to form a transition state resembling an oxocarbenium ion, with the pyridine moiety still in close proximity to the cleaved glycosidic bond. In the final step, this pyridine moiety serves as a base to deprotonate the oxocarbenium ion to form the observed Y ion. The gas phase proton affinity of oxygen atoms in α-D-glycopyranose are calculated to be 204-214 kcal/mol, which is smaller than that of pyridine, 222 kcal/mol. (Stubbs et al., Chem.-Eur. J. 2005, 11, 2651-2659; and Hunter et al., J. Phys. Chem. Ref. Data 1998, 27, 413-656, the entire contents of both of which are herein incorporated by reference.) Alternatively, association of the protonated pyridine with the glycosidic oxygen facilitates a β-hydrogen transfer to oxygen as postulated in Scheme 4 below, which is similar to the reported β-hydrogen transfer of protonated diethylether resulting in loss of ethylene under multiphoton dissociation.
The requirement of acid catalysis involving the labile proton is supported by CID of singly-protonated maltoheptaose derivatized with Girard's T (GT) reagent, a similar aldehyde- and keto-reactive molecule with a fixed-charge quaternary ammonium moiety. In contrast to the protonated charge site of the PRAGS reagent, the inability of this reagent to donate a proton leads to an increase in the CID energy required for a significant extent of fragmentation, and the spectrum is dominated by loss of H2O instead of glycosidic bond cleavage as shown in
Maltoheptaose was employed as a model for linear glycans as it has been well studied by CID, ECD, and EDD mass spectrometry. (Adamson et al, supra; Zhao et al., J. Am. Soc. Mass Spectrom. 2008, 19, 138-150; Kornacki et al., 2012, 23, 2031-2042; Yu et al., Anal. Chem. 2012, 84, 7487-7494, the entire contents of all of which are herein incorporated by reference.) The peak assignments are unambiguous as only Y ions are observed, providing composition and sequence information for the structural analysis of maltoheptaose. This behavior is different from the reported CID of [M+Metal]+, [Mpermethylated+Na]+, [M−H+Cl]2−, and [M−2H]2− glycans, wherein not only Y ions but also A, B, C, X, and Z ions are observed. (Lewandrowski et al., Anal. Chem. 2005, 77, 3274-3283; Zhao et al., supra; Kornacki et al., supra; and Adamson et al., supra. the entire contents of all of which are herein incorporated by reference.) Due to the symmetry of maltoheptaose, multiple pairs of isobaric product ions (B and Z, C and Y, as well as A and X ions) make the assignment ambiguous.
Collisional activation of singly-protonated FRAGS II-derivatized maltoheptaose induces not only C1-O glycosidic bond cleavage but also O-Cx glycosidic bond cleavage and cross-ring cleavages. Many more free radical driven fragmentation pathways in the gas-phase are observed compared with that observed previously in solution. (Duan et al., Glycobiology, 2011, 21: 401-409, the entire contents of which are herein incorporated by reference.) A series of abundant and systematic dissociation patterns including 0,2X, 1,5 X, Z, and n ions are observed and proposed to be driven by hydrogen abstraction followed by rearrangement. As the Z, 1,5X, 0,2X, and n ions are not observed in the CID spectrum of singly-protonated PRAGS-derivatized maltoheptaose, they are proposed to occur via free radical driven mechanisms. In the first step of Z ion formation, the nascent free radical, the carbon-centered radical formed on the FRAGS II reagent, abstracts a hydrogen atom from C5 to generate a carbon-centered radical at this site. In the second step, the resulting radical promotes homolytic cleavage of the glycosidic bond via formation of a double bond between C4 and C5 as shown in Scheme 3.
An alternative mechanism involving hydrogen abstraction from non-reducing terminus C′1-H′1 (anormeric) followed by O—C4 homolytic cleavage and loss of C5-H5 radical is also possible as shown in Scheme 4.
Similar to the formation of Z ions, the formation of 1,5X ions as shown in Scheme 5, 0,2X as shown in Scheme 6, and n ions as shown in Scheme 7, is initiated by hydrogen abstraction by the nascent free radical followed by β-bond cleavages.
Compared with CID of singly-protonated PRAGS-derivatized maltoheptaose, CID of singly-protonated FRAGS II-derivatized maltoheptaose provides much more structural information (
To assess the ability of the PRAGS and FRAGS I reagents to differentiate isobaric glycan structures, the dissociation of several disaccharide isomers was examined. Maltose is a glycan that consists of two glucose bonded through an α1-4 linkage, whereas cellobiose is an isobaric glycan that consists of two glucose bonded through an β1-4 linkage. Lactose differs from cellobiose only in the stereochemistry of C4 in the non-reducing terminus glycan subunit, whereas nigerose differs from maltose and cellobiose only in the linkage type. Collisional activation of these four singly-protonated PRAGS-derivatized disaccharides generates exclusively C1-O glycosidic bond cleavage (forming a Y ion in
The relatively high intensity of the —CH6O3 ion for nigerose compared to the other three isomers may be rationalized by the fact that the hydrogen on C1′ (non-reducing subunit) is highly accessible to the nascent free radical. Clearly, the no ion of cellobiose is significantly more abundant compared to the other three disaccharide isomers. Overall, the difference in fragment ion abundances in the FRAGS CID spectra allows the glycan isomers to be readily distinguished.
Two complex branched glycans, Lewis-Y tetrasaccharide and LNDFH II, were also examined with the new reagents. LNDFH II is employed as a highly branched model glycan, having a reducing terminus in the center and a branch on the N-acetylglucosamine unit, to assess the ability of FRAGS and PRAGS reagents to analyze more complicated glycan structures. Collisional activation of singly-protonated PRAGS-derivatized LNDFH II (
All the fragmentation patterns generated from CID of singly-protonated PRAGS-derivatized LNDFH II can also be found in the CID spectrum of singly-protonated FRAGS II-derivatized LNDFH II (
Lewis-Y tetrasaccharide has an analogous structural feature, and the location of the reducing terminus can be similarly inferred from the related fragmentation patterns (
The proposed mechanism of formation of the Z3HH+Z3HL+H ion is shown in Scheme 10.
The nascent free radical abstracts a hydrogen atom from C1 of one of the branched glycan units. The resulting carbon-centered radical drives the cleavage of the glycosidic bond and formation of a radical on C4 of the N-acetylglucosamine unit, followed by the formation of a double bond between C3 and C4, and finally a second glycosidic bond cleavage to generate the Z3HH+Z3HL+H ion (Scheme 10). The structure of the Z3HH+Z3HL+H ion is confirmed by CID of this characteristic ion as shown in
As depicted below in Scheme 11, resin (Thiopropyl-Sepharose® 6B) is coupled to the FRAGS II reagent.
The synthesis strategy for the Resin-Supported FRAGS II reagent is summarized in Scheme 11 above. The Resin-Supported FRAGS II reagent synthesis was accomplished by benzylic bromination with N-bromosuccinimide (NBS), coupling with TEMPO, hydrolysis of the ester group, activation of the carboxylic acid group, amidation, hydrazinolysis, and finally coupling with Thiopropyl Sepharose® 6B resin. (1 mL, 30 μmol) of resin was washed with deionized water. After the final step, the Thiopropyl Sepharose® 6B resin was suspended in 50% methanol. Five equivalents of the FRAGS II reagent (shown after hydrazinolysis step in Scheme 11) was added to the suspension of the Thiopropyl Sepharose® 6B resin. The reagent and resin suspension was placed on a rocking incubator at room temperature (RT) overnight. After incubation, the resins were washed with 50% methanol, followed by water and 20% ethanol. The resulting resins were stored in 20% ethanol at 4° C. prior to use.
The RS-FRAGS II reagent was coupled with LNFDI and LNFDII. The RS-FRAGS II-derivatized LNFDI and RS-FRAGS II-derivatized LNFDII were treated with dithiothreitol (DTT) to remove the resin support and then subject to ESI and CID by mass spectrometry. The CID spectra for LNFDI and LNFDII using the RS-FRAGS II reagents are shown
Using the RS-FRAGS II reagent, the glycoprotein ribonuclease B (RNase B) was analyzed following the protocol shown schematically in
To gain further insight into the hydrogen atom abstraction processes observed with the FRAGS reagent, C—H and O—H bond dissociation enthalpies (BDEs) of α-1-O-methyl-D-glucopyranose were calculated as described in Example 1. These are displayed graphically in
Glycans were purchased from Sigma-Aldrich (St. Louis, Mo., USA). All solvents are HPLC grade and were purchased from EMD Merck (Gibbstown, N.J., USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA). For desalting, OMIX 100 μL size C-18 tips were purchased from Varian Inc. (Palo Alto, Calif., USA).
To a solution of methyl 5-methylnicotinate (3) (1.51 g, 10 mmol) and N-bromosuccinimide (NBS) (2.13 g, 12 mmol) in CCl4 (50 mL) was added benzoyl peroxide (24.2 mg, 0.1 mmol) under argon. (Lee et al., Analyst 2009, 134, 1706-1712, the entire contents of which is herein incorporated by reference.) The reaction mixture was stirred under reflux for 10 hours. After cooling to room temperature, the reaction mixture was extracted with CH2Cl2 (×3). The extract was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (hexane-EtOAc 1:1) to give methyl 5-(bromomethyl)nicotinate (4) as a white solid (1.17 g, 51%). 1HNMR (500 MHz, CDCl3, δ ppm): 9.13 (d, J=2.0 Hz, 1H), 8.78 (d, J=2.2 Hz, 1H), 8.32 (t, J=2.2 Hz, 1H), 4.50 (s, 2H), 3.96 (s, 3H).); 13C NMR (125 MHz, CDCl3, δ ppm). 28.6, 52.5, 126.0, 133.6, 137.5, 150.4, 153.3, 165.1. ESI-MS: [M+H]+, 230.0, 232.0 (
To a Schlenk flask was added 4 (1.15 g, 5 mmol), (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO, 936 mg, 6 mmol), Cu(OTf)2 (217 mg, 0.6 mmol), copper powder (32.0 mg, 5 mmol), 4,4′-dinonyl-2,2′-bipyridyl (Nbpy, 818 mg, 2 mmol), and benzene (15 mL). (Lee et al., supra.) The reaction mixture was degassed by bubbling argon for 5 min and heated at 80° C. overnight. After cooling the reaction mixture to room temperature, it was filtered through a short pad of silica gel using EtOAc. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography using hexane-EtOAc (3:1). The desired product 5 was obtained as a white solid (1.16 g, 76%). 1HNMR (500 MHz, CDCl3, δ ppm): 9.09 (d, J=2.1 Hz, 1H), 8.70 (d, J=2.0 Hz, 1H), 8.20 (m, 1H), 4.84 (s, 2H), 3.92 (s, 3H), 1.53 (m, 1H), 1.45 (m, 4H), 1.30 (m, 1H), 1.19 (s, 6H), 1.10 (s, 6H); 13C NMR (125 MHz, CDCl3, δ ppm), 16.9, 20.1, 32.7, 39.5, 52.3, 60.0, 75.7, 125.6, 133.4, 135.8, 149.7, 152.5, 165.7. ESI-MS: [M+H]+, 307.3 (
To a solution of 5 (1.07 g, 3.5 mmol) in anhydrous ethanol (20 mL) was added sodium borohydride (266 mg, 7 mmol) portionally under argon. (WO/2008/005457, the entire contents of which are herein incorporated by reference.) The reaction mixture was stirred under reflux overnight. After cooling to room temperature, the reaction mixture was diluted by adding 50 ml EtOAc, washed by water and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (hexane-EtOAc 1:1 to 1:5) to give the desired product 6 as a white solid (731 mg, 75%). 1HNMR (500 MHz, CD3OD, δ ppm): 8.45 (d, J=2.1 Hz, 1H), 8.43 (d, J=2.1 Hz, 1H), 7.83 (m, 1H), 4.89 (s, 2H), 4.68 (m, 2H), 1.66 (m, 1H), 1.54 (m, 4H), 1.38 (m, 1H), 1.26 (s, 6H), 1.16 (s, 6H); 3C NMR (125 MHz, CD3OD, δ ppm), 18.2, 20.8, 33.7, 40.9, 61.4, 62.5, 77.3, 135.5, 136.1, 139.1, 148.0. ESI-MS: [M+H]+, 279.3 (
To a solution of 6 (557 mg, 2 mmol), 2-hydroxyisoindoline-1,3-dione (7, 391 mg, 2.4 mmol), and triphenylphosphine (629 mg, 2.4 mmol) in anhydrous THF (20 mL) was added a solution of diisopropyl azodicarboxylate (DIAD, 525 mg, 2.6 mmol) in 5 ml anhydrous THF at 0° C. under argon. (Deraeve et al., J. Am. Chem. Soc. 2012, 134, 7384-7391, the entire contents of which are herein incorporated by reference.) The reaction mixture was stirred at 0° C. for 30 minutes and then warmed to room temperature and stirred overnight. The reaction mixture was washed by water and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (hexane-EtOAc 1:2) to give the desired product 8 as a white solid (695 mg, 82%). 1HNMR (500 MHz, CDCl3, δ ppm): 8.60 (d, J=2.1 Hz, 1H), 8.58 (d. J=2.0 Hz, 1H), 7.86 (t, J=2.1 Hz, 1H), 7.78 (dd, J1=2.1 Hz, J2=5.5 Hz, 2H), 7.71 (dd, J1=2.1 Hz, J2=5.5 Hz, 2H), 5.22 (s, 2H), 4.83 (s, 2H), 1.55 (m, 1H), 1.45 (m, 4H), 1.32 (m, 1H), 1.26 (s, 6H), 1.15 (s, 6H); 13C NMR (125 MHz, CDCl3, δ ppm), 16.9, 20.1, 32.9, 39.5, 60.0, 75.8, 77.0, 123.5, 128.6, 128.9, 133.5, 134.5, 136.3, 149.5, 149.7, 163.2. ESI-MS: [M+H]+, 424.4 (
To a solution of 8 (1.07 g, 1.5 mmol) in anhydrous ethanol (20 mL) was added hydrazine hydrate (N2H4, 50-60%, 0.86 mL, 15 mmol) portionally under argon. (Deraeve et al., supra.) The reaction mixture was stirred under reflux overnight. After cooling to room temperature, the reaction mixture was concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (CHCl3-Hexane 3:1) to give the desired product 2 as a white solid (378 mg, 86%). 1HNMR (500 MHz, CDCl3, δ ppm): 8.57 (s, 1H), 8.54 (s, 1H), 7.67 (t, J=2.1 Hz, 1H), 4.86 (s, 2H), 4.72 (s, 2H), 1.59 (m, 1H), 1.50 (m, 4H), 1.37 (m, 1H), 1.26 (s, 6H), 1.15 (s, 6H); 13C NMR (125 MHz, CDCl3, δ ppm), 17.0, 20.3, 33.0, 39.6, 60.1, 75.2, 76.2, 132.6, 133.4, 135.3, 148.5, 148.8. ESI-MS: [M+H]+, 294.4 (
O-(pyridin-3-ylmethyl)hydroxylamine (PRAGS, 1) was synthesized following the procedure for synthesis of FRAGS reagent (2). (Deraeve et al., supra.) Overall yield 46%. 1HNMR (500 MHz, CD3OD, δ ppm): 8.54 (d, 1H), 8.47 (dd, J1=1.6 Hz, J2=5.0 Hz, 1H), 7.85 (m, 1H), 7.44 (m, 1H), 4.71 (s, 2H); 13C NMR (125 MHz, CDCl3, δ ppm), 75.4, 125.3, 135.7, 138.4, 149.5, 150.1. ESI-MS: [M+H]+, 125.1 (
1. Attach a C18 pipet tips to a micropipettor and condition twice by aspirating 10 μl of 1:1 acetonitrile/water. 2. Wash pipet tip twice with 10.1 water. 3. Draw up 10 μl of aqueous glycan derivatization solution and return to the main solution. Repeat approximately ten times to saturate the C18 pipet tip with the derivatized glycan. 4. Wash pipet tip twice with 10 μl water. 5. Draw up 10 μl of 1:1 acetonitrile/water and return to the main solution. Repeat approximately ten times to elute glycans. The solution was used directly for ESI-MS.
A Thermo-Fisher Scientific linear quadrupole ion trap (LTQ-XL) mass spectrometer (Thermo, San Jose, Calif., USA) equipped with an electrospray ionization (ESI) source was employed in experiments with PRAGS, FRAGS I and FRAGS II. Derivatized glycan sample solutions were directly infused to the ESI source of the mass spectrometer via a syringe pump at a flow rate of 3 μL/min. Critical parameters of the mass spectrometer include spray voltage of 5˜6 kV, capillary voltage of 30˜40 V, capillary temperature of 275° C., sheath gas (N2) flow rate of 8˜10 (arbitrary unit), and tube lens voltage of 50˜200 V. Other ion optic parameters were optimized by the auto-tune function in the LTQ-XL tune program for maximizing the signal intensity. CID was performed by resonance excitation of the selected ions for 30 ms. The normalized CID energy was 7˜30 (arbitrary unit).
The molecule α-1-O-methyl-D-glucopyranose was used as a simple model system to calculate key bond dissociation enthalpies (BDE) of glycans used in this study, where BDE in this work refers to the bond dissociation enthalpy at 298 K. Initial geometries of the monosaccharide were generated by the MC/MM conformer search with the OPLS 2005 force field using Macromol 8.0 (Schrödinger Inc., Portland, Oreg., USA) implemented in Maestro 8.0 (Schrödinger Inc., Portland, Oreg., USA) under the Linux environment. Within 5 kcal/mol energy, all low energy conformers were initially recorded. After manual screening of obtained structures to avoid redundancy, low energy conformers were selected for further structure optimization by density functional theory (DFT). Each conformer was subject to a geometry optimization using Jaguar 7.5 (Schrödinger Inc., Portland, Oreg. USA) at the B3LYP/6-31+G(d) level. The lowest-energy structure was then utilized as a starting point for optimization of the radical species of interest utilizing DFT at the same level of theory. The single point energy for each species was then refined within Jaguar using B3LYP, M05-2X, and M06-2X density functionals at the 6-311++G(d,p) level of theory with the spin-unrestricted method. The two new generation meta-hybrid functionals other than B3LYP were chosen for their ability to more reliably predict the energetics of organic radical reactions. Thermochemical corrections (zero-point energy and enthalpy) were obtained utilizing the B3LYP/6-311++G(d,p) level of theory and applied to all density functionals for calculation of the bond dissociation enthalpy at 298 K. All calculations were performed using computational resources kindly provided by the Material and Process Simulation Center at the Beckman Institute, Caltech.
BDEs were determined via the isodesmic method, in which the BDE of a reference molecule is utilized to determine the unknown BDE. To determine the BDE of C—H bonds in the monosaccharide, the enthalpy of reaction for hydrogen atom transfer between a carbon-centered methanol radical and each carbon in the sugar was calculated. The use of a reference molecule similar to the compound under study reduces any systematic error from differences between the two species. For the determination of O—H BDEs, an oxygen-centered isopropanol radical was utilized in place of the methanol radical.
As disclosed throughout and evidenced by the data presented in the accompanying figures, for example,
While the present invention has been illustrated and described with reference to certain exemplary embodiments, those of ordinary skill in the art will understand that various modifications and changes may be made to the described embodiments without departing from the spirit and scope of the present invention, as defined in the following claims.
The present application claims priority to and the benefit of U.S. Provisional Application Ser. No. 61/764,975 filed on Feb. 14, 2013, the entire contents of which are incorporated herein by reference.
This invention was made with government support under CHE0416381 awarded by the National Science Foundation. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61764975 | Feb 2013 | US |
