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CHAPTER I
INTRODUCTION
Natural products and mass spectrometry Natural products (also known as secondary metabolites) have always been a significant source of new lead compounds in pharmaceutical industries. About half of the drugs currently in clinical use are natural products or synthetic molecules based on natural product scaffolds.^1 Between 1981 and 2006, 34% of all small molecule new chemical entities were natural products (NP) or natural product derivatives (NPD) and 17% were synthetic molecules whose pharmacophore was from a natural product.^1 In 1999, nine out of the 20 best- selling non-protein drugs were either NPD or developed from natural product leads with combined annual sales greater than $ 16 billion.^2 NP/NPD have various medicinal uses (Figure 1-01) such as antibiotics (e.g., amoxicillin and tigecycline), immunosuppressive agents (e.g., cyclosporine and rapamycin), hypocholesterolemic agents (e.g., lovastatin and pravastatin), antimigraine agents (e.g., sumatriptan and elitriptan), antihypertensive agents (e.g., captopril and enalapril) and anticancer agents (e.g., taxol and calicheamicin).1, 2
Figure 1-01. Structures of drugs currently in clinical use derived from NP.
In traditional MS, purified samples were subjected to high-energy ionization under ultra-high vacuum conditions (“hard” ionization; e.g., electron impact [EI]).^3 The development of electrospray ionization (ESI) MS has marked a milestone in the analysis of natural products. In contrast to earlier ionization techniques such as EI, which were applicable only to thermally stable, low molecular weight volatile compounds, virtually any ion (ranging from inorganic salts to large macromolecules such as proteins) can be analyzed by ESI-MS.^3 Another advantage of ESI-MS over other ionization techniques is that it can be directly coupled to high performance liquid chromatography (HPLC).^3 The sample is sprayed into the ion source as a solution. In the source, the solvent is evaporated under atmospheric pressure in the presence of an electric field, which generates charged ions to be separated by the mass analyzer3, 4^ (Figure 1-02b). In this way, the mass analyzer has become a unique kind of chromatographic detector, able to give information about the molecular mass, the chemical formula and structure of several components of complex biological samples. As a result, the interface of HPLC with ESI-MS has provided an excellent method in the identification and isolation of new secondary metabolites from complex extracts. Typically the first and most important step in the identification of a compound is the determination of its molecular mass. ESI is quite efficient at providing this information. In contrast to “hard” ionization techniques, ESI rarely generates fragments. Molecules are ionized by protonation, cationization, or deprotonation to form pseudomolecular ions.^3 During protonation and
cationization, positively charged ions are formed such as [M+H]+^ and [M+Na]+, respectively. During deprotonation, a proton is removed from the molecule resulting in negatively charged ions [M-H]-. Performing both positive ion and negative ion ESI-MS experiments can give reliable evidence for the molecular weight of a compound. In addition, depending on the mass analyzer used, high accuracy in the determination of the molecular weight can be achieved. From this accurately determined molecular mass (±0.001 mass units), the chemical formula and the number of double bonds, rings or heteroatoms can be inferred. Figure 1-03 illustrates the high resolution and accurate mass measurement of C36-keto- meridamycin in both positive and negative ion mode.^5 The masses and formulae from both ionization modes are in agreement with the expected mass and formula for C36-keto-meridamycin (C 45 H 72 NO 12 , theoretical mass 819.51328). C36-keto-meridamycin is an analogue of the natural product meridamycin produced through genetic manipulation of the meridamycin gene cluster.
Figure 1-03. Mass spectra of C36-keto-meridamycin in both positive (top) and negative (bottom) ion modes.^5
Figure 1-04. Fragmentation pattern of sodiated SCH 27899, a representative of the everninomicin family.^6
Early structural characterization work on the everninomicin family was based on chemical degradation and fast-atom bombardment (FAB) MS.^7 However, since then an ESI tandem MS method has been developed for the structural identification of everninomicins.^6 New members of this class can be discovered based on their isotopic and fragmentation pattern. This is based on the notion that compounds that are structurally similar will have a similar fragmentation pattern. In the case of everninomicins, several characteristic fragment ions are associated with changes on certain functional groups of the molecule (Figure 1-04). For example, a loss of HNO 2 indicates the presence of a nitro group. Also, the loss of ring 2 provides evidence for its presence on sugar ring I. In addition, the characteristic isotopic pattern consistent with the presence of two chlorine atoms in the molecule can aid in identifying this molecules. This very specific fragmentation pattern of everninomicins is extremely valuable in the identification and structural elucidation of new members of this family. MS has found broad application not only in the molecular weight determination and structural elucidation of natural products but also in probing the biosynthetic pathways that lead to their production. Understanding the biosynthetic pathways that form natural products is of immense significance since this information can be used to improve production yields and to synthesize structural analogues via manipulation of the pathway. Feeding studies using isotopic labeled precursors followed by inspection of the labeling pattern in the final product have very frequently been the first step in the investigation of a biosynthetic system. MS has proven to be especially powerful in biosynthetic
Cultures of S. fumanus were grown with Na^15 NO 3 as the sole nitrogen source. Culture extracts were analyzed by MS and were found to contain compounds 1.1 - 1.4 , in which each nitrogen atom was highly enriched (>80%) in (^15) N. Compounds 1.2 - 1.4 (in their 15 N-labeled form) were reintroduced to growing
cultures of S. fumanus to assess their intermediacy in dioxapyrrolomycin biosynthesis. When 15 N-labeled pyrrolomycins C was fed, 1.1 isolated showed 8- 18% incorporation of 15 N at N-1. Tandem MS experiments unambiguously established the regiospecificity of 15 N labeling, as the NO 2 -^ fragment ion generated was entirely at m/z 46, corresponding to the 14 N isotope. The conversion of 1.2 to 1.1 demonstrated that 1.2 is a true intermediate and not just a shunt metabolite of the biosynthesis. In addition, when cultures of S. fumanus were supplemented with 15 N 2 -labeled 1.3 , 15 N 2 -labeled 1.4 was isolated with 86% (^15) N enrichment. Cultures of S. fumanus grown in the presence of 15 N 2 -labeled 1.
produced 15 N 2 -labeled 1.1 with 79% 15 N enrichment, establishing the precursor relationship of 1.4 to 1.. Feeding studies using isotopic labeled methionine were used to study the formation of the methylenedioxy bridge in 1.1 in greater detail. Specifically, in the presence of L-[ methyl -^13 C]-methionine, 1.1 , 1.4 , 1.5 , and 1.6 isolated from cultures of S. fumanus exhibited >85% 13 C enrichment in C-13. These results clearly indicated that the C-13 methyl group of 1.4 , 1.5 , and 1.6 and the C- methylenedioxy group of 1.1 are derived from methionine. Furthermore, feeding of L-[ methyl -^13 CD 3 ]-methionine yielded more information into the formation of the methylenedioxy bridge. The dioxapyrrolomycin ( 1.1 ) isolated had molecular ions
shifted three mass units higher than unlabeled 1.1. Isolated labeled 1.4 was reintroduced to growing cultures of S. fumanus yielding 1.1 enriched in 13 C at the C-13 by 57%. These last two results clearly showed that 1.4 is the ultimate precursor of 1.1 and the transformation of the C-13 methyl group of 1.4 to the methylenedioxy bridge of 1.1 occurs without significant hydrogen-deuterium exchange. These feeding studies enabled the establishment of the biosynthetic precursors of dioxapyrrolomycin and the timing of transformations (nitration, O - methylation, methylenedioxy bridge formation) leading to 1.1 , as outlined in Figure 1-06.^8
Figure 1-06. Biosynthetic pathway of dioxapyrrolomycin as established by stable isotopic feeding studies.^8
Once the intermediates of a natural product’s biosynthesis have been established, they can be used in conjunction with MS to identify and isolate the enzymes involved in the transformations leading to the compound of interest. MS has long been used in the field of enzymology. Unlike other analytical techniques that require the presence of a chromophore or a radioactive label to study an enzymatic reaction, the only requirement for an MS-based method is that the reaction produces a mass change. Most enzymatic reactions, except for racemates and isomerization reactions, do result in a change in molecular mass.
synthase into Escherichia coli and incubated resultant cell lysates with GGDP. Low yields of levopimaradiene required a sensitive detection method to assess the enzyme’s activity. Thus, all reactions were extracted with hexane, concentrated, and analyzed by GC/MS. Identical elution time and fragmentation pattern of biosynthesized levopimaradiene to a synthetic standard verified that the enzyme product was indeed levopimaradiene (Figure 1-07).^9
Figure 1-08. Acyltransferase activity as reported by Pohl et al^10
When the function of an enzyme has been established, further studies involving enzyme mechanism, kinetics and its substrate specificity or flexibility can be undertaken using MS. For example, Pohl and coworkers studied the kinetics and substrate specificity of the acyltransferase activity of a bifunctional enzyme using an MS based assay.^10 In short, the cloned enzyme was incubated in the presence of glucosamine-1-phosphate ( 1.7 ) and acetyl coenzyme A ( 1.8 ) (Figure 1-08). Over time, peaks corresponding to 1.7 ( m/z = 258) disappeared as
new peaks corresponding to N -acetylglucosamine-1-phosphate ( 1.9 , m/z = 300) appeared. Peak quantification with internal standards and calibration curves allowed the calculation of kinetic constants ( K M, k cat, and k cat/ K M) for the reaction. In addition, the substrate flexibility of the enzyme was probed by using truncated analogues of acetyl coenzyme A ( 1.10-1.12 ). The structures of products formed were determined by the same MS method used for enzyme kinetics. The modifications pictured may not deviate vastly form the original structure of acetyl coenzyme A, but could allow the researchers to tag the product using highly specific reactions, such as alkyne/azide coupling.^11 Recent advances in MS instrumentation allow the direct investigation of covalent and non-covalent enzyme intermediates. Anderson and coworkers^12 reported the first direct identification of a hypothesized unstable hemiketal phosphate intermediate bound to the enzyme 3-deoxy-D- manno -2-octulosonate- 8-phosphate (KDO8P) synthase in a noncovalent complex. KDO8P synthase is essential for Gram negative bacteria as it is involved in the lipopolysaccharide biosynthetic pathway. It catalyzes the condensation of arabinose-5-phosphate (A5P), H 2 O, and phosphoenolpyruvate (PEP) to form KDO8P and inorganic phosphate (Pi) (Figure 1-09). It is thought that the mechanism involves labile intermediate I , but this intermediate had never been directly observed before. High resolution ESI-MS interfaced with a novel rapid-mixing technique allowed the detection of the enzyme bound intermediate (E• I ) along with enzyme complexes with each substrate and each product at different ratios as the reaction progressed (Figure 1-10).
system.^14 All covalent intermediates in the pathway have been identified by MS (Figure 1-11).
Figure 1-11. Detection of enzyme-bound intermediates in the yersiniabactin biosynthetic pathway.13, 14
In summary, MS is a powerful technique that can be used at every stage of natural product studies, from discovery and structural characterization of new compounds to biosynthetic enzyme identification and manipulation. This dissertation illustrates the utility of MS in NP investigations by describing its use in two natural product studies: I) K-26, a hypotensive phosphonopeptide metabolite, and II) apoptolidin, an anticancer natural product of polyketide origin.
Case Study I: K-26, a hypotensive phosphonopeptide metabolite Natural and synthetic products containing a carbon-phosphorus (C-P) bond have been found to possess potent biological activities and unique biological roles in primary and secondary metabolism (Table 1-1) and thus, they have been studied thoroughly over the last fifty years. Significant biological activities of C-P containing natural products include herbicidal, antibacterial, antiparasitic, and antihypertensive activities.15,^16 These activities can be attributed to the physical and structural similarity of phosphonic and phosphinic acids to the phosphate ester and carboxylic acid groups present in many biomolecules. In this way, phosphonates can act as substrate or transition state analogs and inhibit enzyme catalysis. In addition, the C-P bond is chemically and thermally highly inert making organophosphonates resistant to chemical hydrolysis, thermal decomposition and photolysis.
Table 1-1. Phosphonates in current use Phosphonate Class Application Cidofovir^1 antiviral cytomegalovirus, HIV Adefovir^1 antiviral hepatitis B, HIV Fosamax^1 antiosteoclastic osteoporosis Fosfomycin^2 antibacterial systemic Fosmidomycin^2 antiparasitic Malaria Bialaphos^2 herbicidal weed control (^1) synthetic compounds 2 natural products
studied C-P bond containing natural products to date is phosphoenolpyruvate mutase (PEP mutase).17-20^ PEP mutase catalyzes the intramolecular rearrangement of phosphoenolpyruvate to phosphonopyruvate (Ppyr). The decarboxylation of Ppyr produces phosphonoacetaldehyde, which is a biosynthetic precursor of AEP, fosfomycin, and bialaphos (Figure 1-13).
Figure 1-13. C-P bond formation by PEP mutase and resulting C-P containing natural products.
Figure 1-14. Possible mechanisms of PEP mutase.
PEP mutase has been studied extensively but the exact catalytic mechanism is still a matter of debate. It has been suggeted that the phosphoryl transfer occurs intramolecularly and with retention of stereochemistry at
phosphorus. There are two possible mechanisms as indicated in Figure 1-14. In mechanism I the phosphoryl group is first transferred to an active site residue of the enzyme and then followed by addition to the C(3) of the pyruvate enol intermediate. Mechanism II involves dissociation of a PO 3 -^ group followed by addition to the C(3) of the pyruvate enol intermediate.17- A second example of a C-P bond forming enzyme is carboxyphosphonoenol pyruvate (CPEP) mutase.21-23^ CPEP mutase catalyzes the formation of the second C-P bond in bialaphos. The two enzymes have 25% amino acid sequence identity and 45% amino acid sequence similarity, suggesting they possess a similar structure and catalytic mechanism.
K-26 ( 1.17 ) from Actinomycete sp. K-26 (NRRL 12379)24-28^ is representative of an uninvestigated class of natural phosphonates which incorporate a phosphonic acid analog of tyrosine. K-26 is reported to possess angiotensin converting enzyme inhibitory activity with an IC 50 value of 12.5 nM, comparable to the widely prescribed antihypertensive drug Captopril. K-26 was initially discovered via ACE bioassay guided fractionation of extracts of Actinomycete sp. K-26. NMR, mass spectrometry, degradation and synthetic studies have demonstrated that K-26 is comprised of N-acetylated L-isoleucine,
