Aryl Sulfate Research Articles

Synthesis and Reactivity of Masked Organic Sulfates.

Nature offers a variety of structurally unique, sulfated endobiotics including sulfated glycosaminoglycans, sulfated tyrosine peptides, sulfated steroids/bile acids/catecholamines. Sulfated molecules display a large number of biological activities including antithrombotic, antimicrobial, anticancer, anti-inflammatory, and others, which arise from modulation of intracellular signaling and enhanced in vivo retention of certain hormones. These characteristics position sulfated molecules very favorably as drug-like agents. However, few have reached the clinic. Major hurdles exist in realizing sulfated molecules as drugs. This state-of-the-art has been transformed through recent works on the development of sulfate masking technologies for both alkyl (sulfated carbohydrates, sulfated steroids) and aryl (sTyr-bearing peptides/proteins, sulfated flavonoids) sulfates. This review compiles the literature on different strategies implemented for different types of sulfate groups. Starting from early efforts in protection of sulfate groups to the design of newer SuFEx, trichloroethyl, and gem-dimethyl-based protection technologies, this review presents the evolution and application of concepts in realizing highly diverse, sulfated molecules as candidate drugs and/or prodrugs. Overall, the newer strategies for sulfate masking and demasking are likely to greatly enhance the design and development of sulfated molecules as non-toxic drugs of the future.

ITC-6102RO, a novel B7-H3 antibody-drug conjugate, exhibits potent therapeutic effects against B7-H3 expressing solid tumors

BackgroundThe B7-H3 protein, encoded by the CD276 gene, is a member of the B7 family of proteins and a transmembrane glycoprotein. It is highly expressed in various solid tumors, such as lung and breast cancer, and has been associated with limited expression in normal tissues and poor clinical outcomes across different malignancies. Additionally, B7-H3 plays a crucial role in anticancer immune responses. Antibody-drug conjugates (ADCs) are a promising therapeutic modality, utilizing antibodies targeting tumor antigens to selectively and effectively deliver potent cytotoxic agents to tumors.MethodsIn this study, we demonstrate the potential of a novel B7-H3-targeting ADC, ITC-6102RO, for B7-H3-targeted therapy. ITC-6102RO was developed and conjugated with dHBD, a soluble derivative of pyrrolobenzodiazepine (PBD), using Ortho Hydroxy-Protected Aryl Sulfate (OHPAS) linkers with high biostability. We assessed the cytotoxicity and internalization of ITC-6102RO in B7-H3 overexpressing cell lines in vitro and evaluated its anticancer efficacy and mode of action in B7-H3 overexpressing cell-derived and patient-derived xenograft models in vivo.ResultsITC-6102RO inhibited cell viability in B7-H3-positive lung and breast cancer cell lines, inducing cell cycle arrest in the S phase, DNA damage, and apoptosis in vitro. The binding activity and selectivity of ITC-6102RO with B7-H3 were comparable to those of the unconjugated anti-B7-H3 antibody. Furthermore, ITC-6102RO proved effective in B7-H3-positive JIMT-1 subcutaneously xenografted mice and exhibited a potent antitumor effect on B7-H3-positive lung cancer patient-derived xenograft (PDX) models. The mode of action, including S phase arrest and DNA damage induced by dHBD, was confirmed in JIMT-1 tumor tissues.ConclusionsOur preclinical data indicate that ITC-6102RO is a promising therapeutic agent for B7-H3-targeted therapy. Moreover, we anticipate that OHPAS linkers will serve as a valuable platform for developing novel ADCs targeting a wide range of targets.

Design and Synthesis of Scopoletin Sulfonate Derivatives as Potential Insecticidal Agents.

(1) Background: Scopoletin and scoparone, two naturally occurring coumarins, have garnered considerable attention and have been introduced to the market in China due to their high insecticidal efficacy and low toxicity. To investigate the structure-activity relationship of these coumarins, a series of scopoletin derivatives with aryl sulfate at C7 and different substitutes at C3 were designed and synthesized, and their insecticidal activity was studied. (2) Methods: A total of 28 new scopoletin derivatives were designed and synthesized. Most target compounds exhibited moderate insecticidal activity against the phytophagous mite Tetranychus cinnabarinus and the brine shrimp Artemia salina. (3) Results: Among these compounds, compounds 5a and 5j possessed the best insecticidal activities against T. cinnabarinus, with LC50 values of 57.0 and 20.0 μg/mL, respectively, whereas that of the control drug was 15.0 μg/mL. Compound 4j exhibited selective insecticidal activities against A. salina, with an LC50 value of 9.36 μg/mL, whereas its LC50 value against T. cinnabarinus was 93.0 μg/mL. The enzymatic inhibitory activity on acetylcholinesterase (AChE) showed a consistent tendency with the insecticidal activity. Further molecular docking analyses predicted the binding conformations of these compounds, which showed a good correlation between the insecticidal activity and the binding scores. (4) Conclusions: In general, a decreased electron cloud density of the Δ3,4 olefinic bond is beneficial for improving the insecticidal activity against both T. cinnabarinus and A. salina. In addition, naphthyl or benzene groups with a sulfate ester at the C7 position could further improve the insecticidal activity against A. salina. AChE was implied to be a site of action for potential insecticidal activity. The results provide insight into the rational design of a new generation of effective coumarin insecticides.

Metabolite Signature of Physical Activity and the Risk of Type 2 Diabetes in 7271 Men.

Large population-based studies investigating the association of physical activity (PA) with the metabolite signature contribute significantly to the understanding of the effects of PA on metabolic pathways associated with the risk of type 2 diabetes. Our study included 8749 Finnish men without diabetes at baseline recruited from the Metabolic Syndrome in Men (METSIM) cohort. We used a questionnaire to measure leisure-time PA. Metabolites were measured in 7271 men as a part of Metabolon’s untargeted Discovery HD4 platform using ultrahigh-performance liquid chromatography–tandem mass spectrometry. We found 198 metabolites significantly associated with PA. Several of these metabolites were novel including especially steroids, amino acids, imidazoles, carboxylic acids, and hydroxy acids. Increased PA was significantly associated with high levels of choline plasmalogens, lysophosphatidylcholines, polyunsaturated fatty acids, carotenoids, long chain acylcarnitines, imidazoles, bilirubins, aryl sulfates, hydroxy acids, indolepropionate, and indolelactate. Several of these metabolites have been previously associated with a decreased risk of type 2 diabetes and with a healthy diet. Our population-based study shows that the metabolite signature of increased PA includes multiple metabolic pathways and is associated with better adherence to a healthy lifestyle.

Fatal Case of Severe Acute Organophosphorus Pesticide Poisoning Complicated with Secondary Hemophagocytic Lympho-Histiocytosis, Severe Lower Intestinal Hemorrhage and Intestinal Mucormycosis

A man in his 50’s, under influence of alcohol, accidentally ingested a pesticide, and was referred for further management and admitted to our hospital. An empty can of Curacron® was found at the site in his farm where he took the alleged pesticide. This raised the suspicion of organophosphorus pesticide poisoning and he was managed at two medical centres before getting admitted to our hospital. His hospital course was complicated with multiorgan dysfunction, shock, respiratory failure and intermediate syndrome. On day five he developed secondary hemophagocytic lymphohistiocytosis (sHLH) and had hematochezia on day six. Colonoscopy revealed multiple circumferential ulcerations in descending and sigmoid colon with luminal narrowing. Biopsy of colonic tissue showed evidence of intestinal Mucormycosis. The clinical presentation of organophosphorus pesticide poisoning in this patient was complicated with multiple issues and included sHLH, chemical gastroenteritis, hemorrhagic ulcers and intestinal zygomycosis. The organophosphorus pesticide ingested by the patient was a 50% emulsifiable concentrate of profenofos along with vegetable oil, soyabean oil and polyglycol ether alkyl aryl sulphate calcium salt 5.25% w/w as an emulsifier/spreading agent. The management of the patient is discussed. Due to the possibility of the emulsifier adhering to the gastrointestinal tract and causing mucosal injury, it is necessary to identify the drug composition and ingredients of the pesticide as soon as possible when managing organophosphorus poisoning.

Isolation and Total Synthesis of Bromoiesol sulfates, Antitrypanosomal arylethers from a Salileptolyngbya sp. Marine Cyanobacterium.

Bromoiesol sulfates A (1) and B (2), new polyhalogenated aryl sulfates, were isolated from a Salileptolyngbya sp. marine cyanobacterium along with their hydrolyzed compounds, bromoiesols A (3) and B (4). To pick up the candidates of their structures, we used Small Molecule Accurate Recognition Technology (SMART), an artificial intelligence-based structure-prediction tool, and their structures were elucidated on the basis of single-crystal X-ray diffraction analysis of bromoiesols (3 and 4). In addition, to verify the structures, the total synthesis of bromoiesol A sulfate (1) and bromoiesol A (3) was achieved. The bromoiesol family, especially bromoiesols (3 and 4), selectively inhibited the growth of the bloodstream form of Trypanosoma brucei rhodesiense, the causative agent of human African sleeping sickness.

Metabolomics profiling for identification of potential biomarkers in chickens infected with avian leukosis virus subgroup J (ALV-J)

There are currently no vaccines or effective drugs to prevent the disorders caused by avian leukosis virus subgroup J (ALV-J). Hence, it is critical to identify potential biomarkers in ALV-J-infected chickens to prevent ALV-J-induced disorders. We hypothesized that ALV-J infection alters metabolic profile in chickens. In the present study, a nontargeted metabolomics approach based on liquid chromatography coupled with mass spectrometry (LC-MS) was used to find differential metabolites in plasma samples from ALV-J-infected chickens and healthy controls. The parametric statistical test (Student's t-test) and fold change analysis were used for univariate analysis. Multivariate statistical analyses included principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The levels of methyl bromide, pyraclonil, hexaflumuron, lythidathion, 3-phosphoglycerol-glutathione, bis-4-nitrophenyl phosphate, 4-ketocyclophosphamide, oxidized photinus luciferin, phenyl sulfate, and aryl sulfate significantly decreased, whereas the levels of 2-methylthiobenzothiazole, irinotecan, methadone, 3-o-ethyl-l-ascorbic acid, and o-acetylneuraminic acid markedly increased in ALV-J-infected chickens as compared to those in healthy controls. These data provide metabolic evidence and potential biomarkers for ALV-J-induced alterations in plasma metabolism.

Assessments of the In Vitro and In Vivo Linker Stability and Catabolic Fate for the Ortho Hydroxy-Protected Aryl Sulfate Linker by Immuno-Affinity Capture Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometric Assay.

Antibody–drug conjugate (ADC) linkers play an important role in determining the safety and efficacy of ADC. The Ortho Hydroxy-Protected Aryl Sulfate (OHPAS) linker is a newly developed linker in the form of a di-aryl sulfate structure consisting of phenolic payload and self-immolative group (SIG). In this study, using two bioanalytical approaches (namely “bottom-up” and “middle-up” approaches) via the liquid chromatography-quadrupole time-of-flight mass spectrometric (LC-qTOF-MS) method, in vitro and in vivo linker stability experiments were conducted for the OHPAS linker. For comparison, the valine-citrulline-p-aminobenzyloxycarbonyl (VC-PABC) linker was also evaluated under the same experimental conditions. In addition, the catabolite identification experiments at the subunit intact protein level were simultaneously performed to evaluate the catabolic fate of ADCs. As a result, the OHPAS linker was stable in the in vitro mouse/human plasma as well as in vivo pharmacokinetic studies in mice, whereas the VC-PABC linker was relatively unstable in mice in vitro and in vivo. This is because the VC-PABC linker was sensitive to a hydrolytic enzyme called carboxylesterase 1c (Ces1c) in mouse plasma. In conclusion, the OHPAS linker appears to be a good linker for ADC, and further experiments would be warranted to demonstrate the efficacy and toxicity related to the OHPAS linker.

Sulfonate Version of OHPAS Linker Has Two Distinct Pathways of Breakdown: Elimination Route Allows Para-Hydroxy-Protected Benzylsulfonate (PHP-BS) to Serve as an Alternative Self-Immolative Group.

Recently we have reported that the ortho-hydroxy-protected aryl sulfate (OHPAS) system can be exploited as a new self-immolative group (SIG) for phenolic payloads. We extended the system to nonphenolic payloads by simply introducing a para-hydroxy benzyl (PHB) spacer. As an additional variation of the system, we explored a benzylsulfonate version of the OHPAS system and found that it has two distinct breakdown pathways, cyclization and 1,4-elimination, the latter of which implies that para-hydroxy-protected (PHP) benzylsulfonate (BS) can also be used as an alternative SIG. The PHP-BS system was found to be stable chemically and in mouse and human plasma, having payload release rates comparable to those of the original OHPAS conjugates.

Introduction of Para-Hydroxy Benzyl Spacer Greatly Expands the Utility of Ortho-Hydroxy-Protected Aryl Sulfate System: Application to Nonphenolic Payloads.

The ortho-hydroxy-protected aryl sulfate (OHPAS) linker is composed of a diaryl sulfate backbone equipped with a latent phenol moiety at the ortho position of one of the aryl units. The Ar-OH released when the ortho phenol undergoes intramolecular cyclization and displaces the second aryl unit can be viewed as a payload. We have shown in the preceding paper that the OHPAS linkers are highly stable chemically and in various plasmas, yet release payloads when exposed to suitable triggering conditions. As an extension of the OHPAS system, we employed a para-hydroxy benzyl (PHB) spacer for coupling to nonphenolic payloads; this tactic again provided a highly stable system capable of smooth release of appended payloads. The PHB modification works beautifully for tertiary amine and N-heterocycle payloads.

Aryl Sulfate is a Useful Motif for Conjugating and Releasing Phenolic Molecules: Sulfur Fluorine Exchange Click Chemistry Enables Discovery of Ortho-Hydroxy-Protected Aryl Sulfate Linker

A new self-immolative linker motif, Ortho Hydroxy-Protected Aryl Sulfate (OHPAS), was devised, and OHPAS-containing antibody drug conjugates (ADC) were tested in vitro and in vivo. Conveniently synthesized using Sulfur Fluorine Exchange (SuFEx) chemistry, it is based structurally on diaryl sulfate, with one aryl acting as a payload and the other as a self-immolative sulfate unit having a latent phenol function at the ortho position. The chemically stable OHPAS linker was stable in plasma samples from 5 different species, yet it can release the payload molecule smoothly upon chemical or biological triggering. The payload release proceeds via intramolecular cyclization, producing a cyclic sulfate coproduct that eventually hydrolyzes to a catechol monosulfate. A set of OHPAS-containing ADCs based on Trastuzumab were prepared with a drug to antibody ratio of ∼2, and were shown to be cytotoxic in 5 different cancer cell lines in vitro and dose-dependently inhibited tumor growth in a NCI-N87 mouse xenograft model. We conclude that OHPAS conjugates will be of considerable use for delivering phenol-containing payloads to tissues targeted for medical intervention.

Abstract LB-A11: Expression of steroid sulfatase induces the integrin signaling pathway in HeLa cells

Abstract Steroid sulfatase (STS) is responsible for the hydrolysis of aryl and alkyl steroid sulfates. STS has a pivotal role in regulating the level of estrogen and androgen responsible for growth of hormone-dependent tumors, such as breast or prostate cancer. However, the molecular function for tumor growth of STS is still not clear. To elucidate possible role of STS on cancer cell proliferation, we investigated whether STS is able to regulate integrin signaling pathway. In this study, we observed that overexpression of STS in HeLa cells induces the expression of integrin β1 and fibronectin, a ligand of integrin α5β1 at protein and mRNA levels. Dehydroepiandrosterone (DHEA), one of the main metabolite of STS, also induces mRNA and protein level of integrin β1 and fibronectin. We found that STS expression and DHEA enhance phosphorylation of focal adhesion kinase (FAK) at tyrosine 925 residue. Moreover, phosphorylation of ERK at threonine 202 and tyrosine 204 residues was also induced, indicating that STS may activate Ras/Raf/MEK signaling pathway. In conclusion, these results suggest that STS expression and DHEA may enhance Ras/Raf/MEK signaling through upregulation of integrin β1 and activation of FAK. Citation Format: Dong-Jin Ye, Yeo-Jung Kwon, Sangyun Shin, Mihye Hong, Hyoungseok Baek, Seung-Ki Ahn, Dongwon Shin, Young-Jin Chun. Expression of steroid sulfatase induces the integrin signaling pathway in HeLa cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A11.

ChemInform Abstract: Aryl Imidazylates and Aryl Sulfates as Electrophiles in Metal‐Free ArSN1 Reactions.

Abstract2,2,2‐Trifluoroethoxy aryl sulfates, formed in situ from imidazolium salts (I), represent a new class of photoactive substrates capable of generating aryl cations which can be trapped by various nucleophiles.

Aryl imidazylates and aryl sulfates as electrophiles in metal-free ArS(N)1 reactions.

Some oxygen-bonded substituents were investigated as leaving groups in photoinduced ArS(N)1 reactions. Irradiation of aryl imidazylates and of the corresponding imidazolium salts mainly caused homolysis of the ArO-S bond. However, previously unexplored trifluoroethoxy aryl sulfates were found to undergo efficient metal-free arylation. The sulfates were conveniently generated in situ by dissolving the corresponding imidazolium salts in basic 2,2,2-trifluoroethanol.

Synthesis of zearalenone-16-β,D-glucoside and zearalenone-16-sulfate: A tale of protecting resorcylic acid lactones for regiocontrolled conjugation.

The development of a reliable procedure for the synthesis of the 16-glucoside and 16-sulfate of the resorcylic acid lactone (RAL) type compound zearalenone is presented. Different protective group strategies were considered and applied to enable the preparation of glucosides and sulfates that are difficult to access up to now. Acetyl and p-methoxybenzyl protection led to undesired results and were shown to be inappropriate. Finally, triisopropylsilyl-protected zearalenone was successfully used as intermediate for the first synthesis of the corresponding mycotoxin glucoside and sulfate that are highly valuable as reference materials for further studies in the emerging field of masked mycotoxins. Furthermore, high stability was observed for aryl sulfates prepared as tetrabutylammonium salts. Overall, these findings should be applicable for the synthesis of similar RAL type and natural product conjugates.

Aryl Sulfotransferase from Haliangium ochraceum: A Versatile Tool for the Sulfation of Small Molecules

AbstractSulfation is an important molecular modification that regulates essential cellular processes and is also implicated in numerous pathological processes. The enzymes responsible for this reaction in living organisms are sulfotransferases. The gene Hoch_5094 from Haliangium ochraceum is annotated as a putative sulfotransferase. The arylsulfotransferase codified by this gene (HocAST) was expressed heterologously in E. coli and showed aryl sulfotransferase activity. Circular dichroism analysis of HocAST showed a main α/β secondary structure that agrees with the overall structure of other cytosolic sulfotransferases. Interestingly, HocAST was able to use both p‐nitrophenyl sulfate and 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) as sulfuryl donors contrary to that of aryl sulfate sulfotransferase, which cannot use PAPS as a donor. Regarding the specificity towards the acceptor, HocAST has shown quite a wide scope and was able to accept several mono‐ and dihydroxylated phenols and other phosphorylated compounds as substrates.

Experimental and Theoretical Insights into the Mechanisms of Sulfate and Sulfamate Ester Hydrolysis and the End Products of Type I Sulfatase Inactivation by Aryl Sulfamates

Type I sulfatases catalyze the hydrolysis of sulfate esters through S-O bond cleavage and possess a catalytically essential formylglycine (FGly) active-site residue that is post-translationally derived from either cysteine or serine. Type I sulfatases are inactivated by aryl sulfamates in a time-dependent, irreversible, and active-site directed manner consistent with covalent modification of the active site. We report a theoretical (SCS-MP2//B3LYP) and experimental study of the uncatalyzed and enzyme-catalyzed hydrolysis of aryl sulfates and sulfamates. In solution, aryl sulfate monoanions undergo hydrolysis by an S(N)2 mechanism whereas aryl sulfamate monoanions follow an S(N)1 pathway with SO2NH as an intermediate; theory traces this difference to the markedly greater stability of SO2NH versus SO3. For Pseudomonas aeruginosa arylsulfatase-catalyzed aryl sulfate hydrolysis, Brønsted analysis (log(V(max)/K(M)) versus leaving group pK(a) value) reveals β(LG) = -0.86 ± 0.23, consistent with an S(N)2 at sulfur reaction but substantially smaller than that reported for uncatalyzed hydrolysis (β(LG) = -1.81). Common to all proposed mechanisms of sulfatase catalysis is a sulfated FGly intermediate. Theory indicates a ≥26 kcal/mol preference for the intermediate to release HSO4(-) by an E2 mechanism, rather than alkaline phosphatase-like S(N)2 substitution by water. An evaluation of the stabilities of various proposed end-products of sulfamate-induced sulfatase inactivation highlights that an imine N-sulfate derived from FGly is the most likely irreversible adduct.

Catalytic Proficiency: The Extreme Case of S–O Cleaving Sulfatases

As benchmarks for judging the catalytic power of sulfate monoesterases, we sought to determine the rates of spontaneous hydrolysis of unactivated alkyl sulfate monoesters by S-O bond cleavage. Neopentyl sulfate proved to be unsuitable for this purpose, since it was found to undergo hydrolysis by a C-O bond cleaving mechanism with rearrangement of its carbon skeleton. Instead, we examined the temperature dependence of the spontaneous hydrolyses of aryl sulfate monoesters, which proceed by S-O cleavage. Extrapolation of a Bronsted plot [log(k(25)(N)) = (-1.81 ± 0.09) pK(a)(LG) + (3.6 ± 0.7)] based on the rate constants at 25 °C for hydrolysis of a series of sulfate monoesters to a pK(a)(LG) value of 16.1, typical of an aliphatic alcohol, yields k(25)(N) = 3 × 10(-26) s(-1). Comparison of that value with established k(cat) values of bacterial sulfatases indicates that these enzymes produce rate enhancements (k(cat)/k(uncat)) of up to 2 × 10(26)-fold for the hydrolysis of sulfate monoesters. These rate enhancements surpass by several orders of magnitude the ~10(21)-fold rate enhancements that are generated by phosphohydrolases, the most powerful biological catalysts previously known. The hydrolytic rates of phosphate and sulfate monoesters are compared directly, and the misleading impression that the two classes of ester are of similar reactivity is dispelled.

Abstract 1014: Induction of human steroid sulfatase by TNF-α through PI3-K/Akt signaling pathway in PC-3 human prostate cancer cells

Abstract Steroid sulfatase (STS) is responsible for the hydrolysis of aryl and alkyl steroid sulfates and has a pivotal role in regulating the formation of biologically active estrogens. STS may help support the growth of hormone-dependent cancers, including prostate cancers and is considered as a new promising drug target for treating estrogen-mediated cancer. However, the molecular mechanism of STS expression is still not well-known. Previously, cytokines such as TNF-α and IL-6 were known to increase the activity of STS but the changes in expression have not been completely elucidated. To investigate whether cytokines are able to regulate transcription of STS gene, we studied the effect of TNF-α on STS expression in human prostate cancer PC-3 cells. Western blot and RT-PCR analyses showed that treatment with TNF-α significantly induced expression of mRNA level and protein level of STS in concentration- and time-dependent manners. Treatment with TNF-α resulted in a strong increase in the phosphorylation of Akt on Ser 473 and when the cells were treated with PI3-K inhibitors such as LY294002 or wortmannin or Akt inhibitor (Akt inhibitor IV) together, STS expression induced by TNF-α was significantly blocked. Moreover, inhibition of Akt activation by transfecting dominant-negative Akt plasmid also prevented TNF-α -mediated STS gene expression. Taken together, these data strongly suggest that TNF-α induces STS expression through PI3-K/Akt signaling pathway in PC-3 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1014. doi:10.1158/1538-7445.AM2011-1014

Heparin/Heparan Sulfate N-Sulfamidase from Flavobacterium heparinum

Sulfated polysaccharides such as heparin and heparan sulfate glycosaminoglycans (HSGAGs) are chemically and structurally heterogeneous biopolymers that that function as key regulators of numerous biological functions. The elucidation of HSGAG fine structure is fundamental to understanding their functional diversity, and this is facilitated by the use of select degrading enzymes of defined substrate specificity. Our previous studies have reported the cloning, characterization, recombinant expression, and structure-function analysis in Escherichia coli of the Flavobacterium heparinum 2-O-sulfatase and 6-O-sulfatase enzymes that cleave O-sulfate groups from specific locations of the HSGAG polymer. Building on these preceding studies, we report here the molecular cloning and recombinant expression in Escherichia coli of an N-sulfamidase, specific for HSGAGs. In addition, we examine the basic enzymology of this enzyme through molecular modeling studies and structure-function analysis of substrate specificity and basic biochemistry. We use the results from these studies to propose a novel mechanism for nitrogen-sulfur bond cleavage by the N-sulfamidase. Taken together, our structural and biochemical studies indicate that N-sulfamidase is a predominantly exolytic enzyme that specifically acts on N-sulfated and 6-O-desulfated glucosamines present as monosaccharides or at the nonreducing end of odd-numbered oligosaccharide substrates. In conjunction with the previously reported specificities for the F. heparinum 2-O-sulfatase, 6-O-sulfatase, and unsaturated glucuronyl hydrolase, we are able to now reconstruct in vitro the defined exolytic sequence for the heparin and heparan sulfate degradation pathway of F. heparinum and apply these enzymes in tandem toward the exo-sequencing of heparin-derived oligosaccharides.