Biocatalytic Strategies Towards [4++2] Cycloadditions

Benjamin R. Lichman,[b, c] Sarah E. O’Connor,[b] and Hajo Kries*[a]

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1. Introduction
Biocatalysis and synthetic organic chemistry play by the same physicochemical rules, but their strong suits are fundamentally different. A case in point are cycloaddition reactions: their wide use in synthetic organic chemistry contrasts with their scarcity in biological systems. When Diels and Alder first discovered synthetic [4+2] cycloadditions they proposed that these reactions may be responsible for the formation of natural products;[1] this prediction was accurate, but enzymes performing cycloadditions remained elusive for many decades.

Remarkably, Diels-Alderases raised from antibodies in the laboratory preceded isolation of natural enzymes by several years.[2] Although Diels–Alder reactions had been implicated in biosynthetic pathways since at least the 1970s,[3] it took until the mid-1990s for the first observation of natural enzymatic activity catalysing a Diels–Alder reaction.[4] In recent years, a“gold rush” for enzymatic cycloadditions has unveiled a number of these biosynthetic transformations. However, the natural reactions play a different role to those employed by synthetic chemists. While synthetic chemists often harness in termolecular cycloadditions as a retrosynthetic disconnection, the natural examples tend to happen intramolecularly and occur late in the biosynthetic pathway.

In this review, we will explore the catalytic mechanisms of natural and unnatural [4+2] cyclases and compare them to chemical strategies. Determining accurate mechanisms of enzyme catalysed cycloadditions has proved to be challenging. The atom connectivity of reactants and products can readily be used to qualify a reaction as a formal [4+2] cycloaddition, but considerable debate concerns the degree of synchronicity[5] which is often taken as a criterium for “true” Diels–Alder reactions. As intriguing and important as the mechanistic study of enzymatic cycloadditions is, knowing the synchronicity of bond-formation events is not a priority for biocatalytic applications. Hence, we will cover enzymes catalysing formal[4+2] cycloadditions whether or not the detailed mechanism is known. This includes Diels–Alderases catalysing a concerted[4+2] cycloaddition and enzymes employing stepwise Michaelaldol mechanisms. We will consider [4+2] cyclases to be any enzyme catalysing a reaction in which reactants with four and two atoms are connected to form a cycle of six while undergoing a reduction in bond multiplicity.[6,7] Compared to the excellent reviews of the past years covering pericyclases,[7–14] here we also highlight the newest discoveries in plants and focus particularly on the catalytic devices of the natural enzymes, designer enzymes and small molecule catalysts.

[a] Dr. H. Kries
Independent Junior Research Group
Biosynthetic Design of Natural Products
Leibniz Institute for Natural Product Research and Infection Biology
Hans Knçll Institute (HKI Jena)
Beutenbergstr. 11a, 07745 Jena (Germany)
E-mail: [email protected]

[b] Dr. B. R. Lichman, Prof. Dr. S. E. O’Connor
Department of Biological Chemistry
The John Innes Centre, Colney Lane, Norwich (UK)

[c] Dr. B. R. Lichman
Current address: Department of Biology
University of York, York, YO10 5YW (UK)
The ORCID identification number(s) for the author(s) of this article can be
found under: https://doi.org/10.1002/chem.201805412.

2. How chemists accelerate [4+2] cycloadditions
Although chemists have often turned to nature for inspiration, small-molecules catalysing cycloadditions were synthesised long before biocatalysts for these reactions were designed or discovered. Chemical catalysis of Diels–Alder reactions, for example, with simple Lewis acids such as aluminum trichloride, can achieve rate accelerations of 105 fold; the same range as typical, moderately efficient enzyme catalysts.[15] This spectacular effect powers asymmetric catalysts that accelerate and direct the Diels–Alder reaction into a single stereochemical outcome.[16,17] The Lewis acid metal centre of these catalysts provides the core catalytic effect, whilst sterically demanding ligands close off undesired routes by favouring specific orientations of the reactants. Electronically, cycloaddition catalysis has
been explained by a narrowing of the energy gap between the frontier orbitals of the diene and, through binding of the Lewis acid, the more electron-depleted dienophile.[5,18]

For biological cyclases to become relevant in industrial biocatalysis, they must be able to compete with enantioselective chemical catalysts. These chemical catalysts have been meticulously designed for high catalytic efficiency and enantioselectivity and thus provide a rigorous reference point for discussing their biological counterparts (Scheme 1). Corey’s C2 symmetric aluminum diamine 1 is illustrative of the chemo-catalytic approach.[19] Here, the aluminum metal centre is coordinated by the dienophile with the less hindered lone pair opposite

the oxazolidinone moiety. p–p interactions likely govern the preference of the vinyl residue to point towards the phenyl group of the catalyst and the cyclopentadiene attacks with endo preference from the less shielded face. As a result, the cycloaddition product 2, an intermediate in the total synthesis of prostaglandin, is formed with 96% enantiomeric excess. Conformational restriction of the dienophile is crucial for the success of enantioselective catalysts that use only monodentate coordination, such as the aluminum diamines. In Corey’s oxazaborolidine ligand complex 3, a weak interaction with the
C-alpha hydrogen of the ketone ligand has been proposed to account for this restriction, furnishing excellent enantioselectivities up to 99% ee.[19] Evan’s cationic copper(II) bisoxazolines are noteworthy because copper(II) complexes are relevant for the construction of artificial metalloenzymes.[20] Due to the bidentate coordination of the dienophile and the C2 symmetry at the distorted square-planar copper centre in complex 4, predictions of the transition state are straightforward, and it follows that facial selectivity of the dienophile is controlled by the bulky tert-butyl groups.[21]

For cycloadditions featuring hydrophobic reactants, for example, the Diels–Alder reaction of methylvinylketone and cyclopentadiene, solvent selection can be influential. In a polar solvent like water, greasy reactants will associate with each other, and this hydrophobic proximity effect can accelerate the Diels–Alder reaction by two to three orders of magnitude.[22] To achieve rate accelerations, an enzyme in aqueous solution must therefore provide more than the hydrophobic effect in solvent alone. A more exotic catalytic device not directly available to enzymes are high pressures of several thousand atmospheres, which have been useful in mechanistic studies and total synthesis.[23,24] High pressures drive formation of the, relative to the reactants, more compact Diels–Alder transition state.[25]

Enantioselective catalysts often show high stereoselectivity like enzymes but compare poorly in terms of catalytic efficiency: reactions typically take days to reach completion. For copper bisoxazolinones, dissociation constants in the high millimolar range have been reported.[26] Given the open and solvent exposed arrangement of substrates on the metal centre

dency to accommodate hydrophobic molecules on its surface(Scheme 1 B).[27] Upon protein addition, the high endo/exo selectivity of the catalyst (96:4) was maintained while the chiral protein environment conferred a 93% enantiomeric excess. The catalytic rate, however, dropped slightly compared to the free ligand. With 2 mol% catalyst loading of a 66 kDa protein, the protein exceeded the product mass by far, underlining the challenge to create designer biocatalysts with fast catalytic rates (i.e. kcat) and high turnover numbers (TON).

With protein catalysts comes the promise of genetic fine tuning and metalloenzymes are no exception to this.[28,29] Enzyme binding pockets enclose substrates with a shell of protein side-chains that can be mutated to a vast variety of shapes and decorated with charges and H-bonding donors and acceptors. Bos and colleagues installed a conjugation site for bromoacetamide ligands by introducing the mutation Met89Cys into the small, homodimeric Lactococcal multidrug resistance Regulator (LmrR) protein (Scheme 1 B).[30] By conjugating a phenanthroline ligand and adding copper, an enantioselective catalyst was created reaching a good enantiomeric excess of 97% (endo/exo=95:5). In this case, conjugation to LmrR not only rendered the reaction enantioselective but also accelerated it, as indicated by a 4.7-fold increase in yield after three days reaction time. The active site was responsive to mutation which could perhaps be exploited by more comprehensive mutational screens.

Metallo-cyclases appear to provide the best of both worlds for catalyst design: catalysis is provided by the metal centre and selectivity by the protein. This separation of roles may ultimately make these systems easier to control and modify than natural cyclases, in which catalysis and selectivity are intimately coupled within the protein structure. The presence of a protein scaffold, amenable to mutation and directed evolution,[31] makes these synthetic enzymes a promising prospect for industrial biocatalysis. Despite the abundance of natural enzymes with catalytic metal centres, and the utility of metals in
cycloaddition catalysis, natural [4+2] cyclases utilising metal catalysis have not been observed. Instead, enzymes using other catalytic strategies have been discovered.

4. Pulling the trigger
Biosynthesis is a system replete with reactive intermediates and cascade-like multiple transformations. This has led to different strategies for catalysing cycloadditions which take advantage of multiple, cascaded reaction steps. For example, enzymes have been discovered that couple cyclisation to redox steps, a reduction or oxidation step effectively “pulling the trigger” by creating a reactive intermediate primed to undergo subsequent cyclisation.

4.1. Oxidation: solanapyrone synthase
Solanapyrone synthase (SPS) catalyses an NAD+ dependent oxidation prior to [4+2] cyclisation (Scheme 2 A). The crude enzyme was isolated from the fungal pathogen Alternaria solani and shown to catalyse the exo-selective formation of the

decalin moiety found in the solanapyrones. In the absence of the cell-free extract, the aldehyde prosolanapyrone III (6) spontaneously cyclises to form a mixture of the exo and endo adducts 7 and 8 in a 3:97 ratio. In contrast, the crude enzyme selectivity was 87:13.[4] The crude enzyme was also shown to perform kinetic resolution of the cyclised (:)-solanapyrone B through enantioselective oxidation to (@)aldehyde solanapyrone A (7). This stereoselectivity indicates recognition of the decalin by the oxidase and perhaps coupling of the oxidation and cyclisation steps.

When the solanapyrone biosynthetic gene cluster was identified and cloned, Sol5, a flavin-dependent oxidase (SPS), was determined to be responsible for both the oxidation and cyclisation reactions.[32] The purified, bifunctional enzyme can convert the alcohol substrate prosolanapyrone II (5) to solanapyrone via a two-step oxidation–cyclisation reaction with high enantioselectivity (99% ee). Notably, the cycloaddition activity of the enzyme was observed to denature more rapidly than the oxidation activity, suggesting there may be a structural element specific to cyclisation.[33] Oxidation of the hydroxymethyl in prosolanapyrone II (5) lowers the LUMO of the dienophile, which promotes the cycloaddition. Further catalytic interactions with Sol5 to promote cyclisation have not yet been identified. To account for the perfect enantiomeric purity, the enzyme must control cyclisation, though the presence of 13% endo-product 8 suggests imperfect enzyme selectivity.

4.2. Reduction: iridoid synthase
Reduction, too, can pave the way for a cyclisation reaction, as exemplified by iridoid synthase (ISY). Iridoid monoterpene biosynthesis in plants starts with the reductive cyclization of 8-oxogeranial 9 to nepetalactol (Scheme 2 B). Through an accumulation of evidence, it has become apparent that the first enzyme in this process, ISY,[34–37] catalyses the stereoselective 1,4-reduction of 8-oxogeranial 9, but not subsequent cyclisation.

After forming the uncyclised and reactive enolate via reduction, ISY does not catalyse its cyclisation, but appears to release it as a product. This intermediate then cyclises or unproductively tautomerises outside the ISY active site. Hence, in conversion assays with CrISY (Catharanthus roseus) and 8-oxogeranial 9, small amounts of multiple reaction products are consistently observed, in addition to the expected bicyclic (7S) cis-trans-nepetalactol 11. These include monocyclic iridodials with varying bridge stereochemistry and the uncyclised 8-oxocitronellal 12.[38] Strikingly, the proportion of diastereomeric products formed is independent of the ISY employed, even in the case of AmISY (Antirrhinum majus, snapdragon) which produces the 7R product series in contrast to the 7S series produced by CrISY (Scheme 2 C).[38] Instead, the distribution of products is affected by conditions such as buffer type and concentration, with increased buffer concentrations resulting in greater proportions of side-products. These results indicated that the cyclisation occurs in the solvent, with the buffer acting as a general acid catalyst.[39]

The crystal structures of CrISY co-crystallised with cofactor and substrate or inhibitor support this: the substrate binds in a linear conformation conducive to reduction but not cyclisation.[40–42] Investigation into the cyclisation propensity of substrate analogues suggested nepetalactol formation occurs by a stepwise Michael addition, and not Diels–Alder cyclisation(Scheme 2 B);[43] it now appears these experiments were probing not the enzymatic but the general-acid/buffer catalysed mechanism. Although it has become clear that iridoid synthase does not control the cyclisation step, the abundance of certain nepetalactol isomers in some plants led to the hypothesis that stereocontrol may be provided by a second enzyme, which will be discussed in the next section.

5. Controlling (stereo)selectivity
Molecular complexity is generated at cycloaddition steps, and through controlling the chemo and stereoselectivity of these steps diverse products can be obtained. Hence, in some biosynthetic pathways, cycloaddition steps constitute crucial metabolic branch points. Interestingly, there are multiple examples where closely related enzymes appear to catalyse divergent selectivities.

5.1. Nepetalactol-related short-chain reductases
We have recently identified three enzymes from the catmint Nepeta mussinii which control formation of nepetalactol, precursor to the cat attractant nepetalactone.[36,39] These enzymes, the nepetalactol-related short-chain-dehydrogenase/reductases(NEPS), appear to be specific to the Nepeta genus and are putative [4+2] cyclases. The activities of these NAD-dependent enzymes are revealed in cascade reactions with 8-oxogeranial 9, ISY and appropriate cofactors. NEPS1 seems to be a bifunctional cyclase and dehydrogenase, promoting formation of cistrans-nepetalactol 11 at the expense of side products and oxidising it into nepetalactone (Scheme 2D). With NEPS2, only cis–trans cyclase activity was found. NEPS3 has the most interesting activity because it overrules the non-enzymatic cyclisation selectivity and promotes formation of cis-cis-nepetalactol 13, an isomer formed in only trace amounts without enzyme.

Despite the presence of the nicotinamide co-factor, none of the NEPS enzymes catalyse the ISY reaction, reduction of 8-oxogeranial 9. Although they can accept 8-oxocitronellal 12 as a substrate, this occurs with poor yields and only in the presence of buffer. Altogether, the data indicate that NEPS1-3 accept 8-oxocitronellyl enol, the short-lived product of ISY, as a substrate and perform cyclisation in a redox-neutral manner. Based on structural and mutational analysis of NEPS1 and NEPS3, it appears that whilst both enzymes provide selectivity in the [4+2] cyclisation, they do so via different mechanisms.
NEPS1-catalysed cis-trans cyclisation appears robust to mutation, and the enzyme may merely be protecting the reactive intermediate from the solvent, preventing the formation of side products. NEPS3, on the other hand, must be actively binding its substrate in a specific conformation to yield the cis–cis isomer. A similar, cis–cis selective enzyme might work together with AmISY in antirrhinoside biosynthesis(Scheme 2 C). NEPS3 interactions with the reactive intermediate appear to be controlled by oxyanion interaction with Ser154, though further structural and computational data are required
to fully understand this cyclase’s mechanism.

5.2. Catharanthine and tabersonine synthase
The biosynthesis of the cancer drug vinblastine in the plant Catharanthus roseus provides a remarkable example in which a single precursor (dehydrosecodine [15]) is cyclized to two different products (catharanthine [16] and tabersonine [17]) by two chemo and stereoselective [4+2] cyclases (Scheme 3A). Decades of biomimetic chemical investigation[44–47] support the notion that divergent Diels-Alder cyclisations of the hypothetical intermediate dehydrosecodine (15) could provide the two distinct scaffolds, catharanthine (16) and tabersonine (17), by an inverse- and a normal-electron-demand Diels–Alder reaction, respectively. Through a combination of in planta silencing, bioinformatics and recombinant assays, the enzymes controlling this crucial branch point have finally been identified.[48,49] Two closely related a/b-hydrolase enzymes with 78% sequence identity, catharanthine synthase (CS, HL1) and tabersonine synthase (TS, HL2), act as divergent, stereoselective cyclases. The role of CS and TS was indicated by gene silencing in planta and then verified by reconstitution of the pathway in vitro and in planta.

The enzymes CS and TS have been shown to control the stereoselectivity of a [4+2] cyclisation reaction, but their identity as “dedicated” Diels–Alderases can only be confirmed when their substrate is known: to date, the cyclisation activity has only been observed as a part of the multi-enzyme cascade. The dehydrosecodine intermediate (15) remains the most likely candidate for the Diels-Alder step, but the divergent reactions would proceed via specific transition states. Selectivity may be achieved by steric effects, that is, controlling the conformation of the substrate in the active site, or electronic effects, that is, lowering the LUMO to reduce the transition state energy. The latter possibility is intriguing as the reactions have different electronic characteristics (inverse-electron-demand versus normal-electron-demand) so the substrate would require different stabilising interactions with the enzymes’ active sites.

Structural analysis of the enzymes could provide insight into the mechanism of these fascinating cyclases. 5.3. Decalins in fungal polyketides Similar to the aforementioned solanapyrone synthase Sol5
(Section 4.1.), several fungal [4+2] cyclases transform the polyunsaturated products of partially reducing, modular polyketide synthases into decalins.[50–55] Most of the precursor molecules, in contrast to prosolanapyrone II (5), carry an activating carbonyl in the dienophile already in the correct oxidation state. For instance, enzymes LovB and LovC from the lovastatin producing Aspergillus terreus were attributed to a pathway including a decalin-forming stereoselective Diels–Alder step(Scheme 3B).[56] LovB is an iterative polyketide megasynthase with multiple subunits, whilst LovC is a separate accessory protein which catalyses a key enoyl reduction step in the biosynthesis. In the absence of LovC, purified LovB forms pyrone products. The LovC protein is proposed to complex strongly to LovB. With substrate analogue 18 probing the Diels–Alder step, LovB shifts the product outcome in favour of the lovastatin-like product 19, but only to an underwhelming fraction of 4%. The substrate analogue might not be ideal, or the nonenzymatic reaction dominates the outcome. The proposed transition-states of the Diels–Alder products show that for both the spontaneous products, the C-6 methyl is positioned in a pseudo-equatorial position, whilst this methyl would occupy a more hindered pseudo-axial position in the enzyme product.

To achieve this selectivity, LovB would have to confine the ligand into the correct conformation for the product to form, possibly through van-der-Waals and steric interactions. A family of enzymes unrelated to Sol5 or LovB forms decalins in fungal polyketide-nonribosomal peptide hybrids.[50,51,53–55,57] Astonishingly, homologous [4+2] cyclases Fsa2[55] and Phm7 generate the decalin natural products equisetin (20) and phomasetin (21) with opposite configurations at all chiral centres (Scheme 3 C).[51] Both trans-decalin configurations differ from the spontaneous cis-decalin product. When integrated into a Phm7 knock-out strain, Fsa2 also acted on the slightly longer phomasetin precursor 22 with equisetin chirality, demonstrating the potential of decalin synthases for pathway engineering.[51] As purified protein, Fsa2 was used in the chemoenzymatic synthesis of equisetin (20) with perfect stereoselectivity which chemical cyclisation failed to achieve.[57]

Relatively high kcat values measured both for Fsa2 (5.8 s@1)[57] and related MycB (0.9 s@1)[53] certainly benefit such biocatalytic applications. Another homolog, PvhB, has recently been shown to use
“carboxylative deactivation” as a strategy directly opposed to the triggering reactions discussed in Section 4.[58] By installing a carboxylate conjugated with the diene, nonenzymatic formation of the trans-decalin is suppressed and PvhB promotes generation of the cis-decalin instead.

6. Controlling reaction trajectories
Engineering or designing enzyme activity is most effective when the mechanism is well understood. A full understanding of mechanism ideally involves a combination of experimental and computational analyses, and the two enzymes described in this section, SpnF and LepI, have been investigated using both approaches. Computational analysis of their mechanisms suggests both enzymes catalyse reactions across an energy landscape featuring multiple possible mechanisms and ‘ambimodal’ transition states, in which the reaction trajectory bifurcates to two different products. However, in both cases, a
single product is ultimately preferred—a phenomenon compellingly referred to asacase of “all roads lead to Rome”.[59]

6.1. Spinosyn A bioynthesis
SpnF, from spinosyn A biosynthesis (Scheme 4 A), catalyses a transannular [4+2] cycloaddition, contributing a rate enhancement of approximately 500-fold (kcat=14 min@1 vs. knon=0.03 min@1). SpnF does not alter the stereochemical course of the reaction: the non-enzymatic and SpnF-catalysed reactions have identical stereochemical outcomes.[60] Mechanistic analyses of the methyltransferase homologue SpnF are based on a structure with an SAH co-factor, but no other ligand bound. The role of SAH appears to be primarily structural, and accordingly, mutations in the cofactor binding
region typically result in denaturation.[60, 64] There have been extensive computational investigations into the SpnF-catalysed reaction utilising multiple approaches(Scheme 4 B).[61–63,65–68] These simulations are based on the SpnF crystal structure and computational docking[64] and show multiple mechanisms in the gas-phase leading to the [4+2] product 23. Alongside the direct route via a concerted [4+2] transition state, Patel et al.[67] proposed an alternative route via[6+4] intermediate 24 which undergoes a Cope rearrangement to reveal the [4+2] product 23. Furthermore, they propose
that the initial transition state is ambimodal and able to lead to either [4+2] or [6+4] adducts.

Three groups found no evidence of the [6+4] mechanism in simulated, enzyme-catalysed reaction trajectories (Scheme 4 B). Zheng and Thiel[62] propose that rate enhancement was primarily due to an H-bond between Thr196 and the C15 carbonyl, causing electron density withdrawal and increased reactivity of the dienophile. They also suggest that the enzyme favours adoption of the s-cis C5@C6 substrate diene conformation necessary for cycloaddition, possibly aided by Trp256 H-bonding the C1 carbonyl. In addition to these factors, Chen et al.[61] propose p–p stacking interactions between the substrate and residues Tyr23 and Trp256 which help position the substrate into a reactive conformation.
The [4+2] mechanism suggested by Yang et al.[63] is remarkably different in the proposed nature of catalysis (Scheme 4 B).

The Thr196 C15 carbonyl H-bond is absent as the transition state preferentially forms an intramolecular interaction. Instead, H-bonds are present between the transition state and His42, Glu152 and Trp256, decreasing the reaction energy barrier. Most remarkably, the authors suggest that the trajectory over the ambimodal transition state is determined by kinetic energy transfer from hydrophobic residues (Val26, Leu30 and Leu198) to the substrate via femtosecond scale vibrational collisions.

This radically dynamic view of enzyme catalysis could be significant in enzymes beyond Diels-Alderases. Investigation into the enzyme via kinetic isotope effects supports aspects of the computational mechanisms[69] such as the asynchronous formation of the C7@C11 bond prior to the C4@C12 bond.[65,67] Mutational analysis of SpnF could verify the proposed roles of active site residues, but unfortunately such experiments have so far been hindered by a lack of enzyme stability.[60,64] Other experimental targets may include solving structures with substrate or substrate analogues bound and
determining definitively whether SAM or SAH is the in vivo cofactor. Investigating the presence of the [6+4] mechanism is experimentally challenging as the Cope rearrangement will always lead to the [4+2] product 23. Design of novel substrates may allow this fascinating proposition to be examined.

6.2. Leporin C biosynthesis
A remarkably similar ambimodal pericylic transition state has also been proposed for the LepI cyclase, though in this instance the energy landscape, which includes a stable intermediate, allows the system to be more experimentally tractable.[70] The biosynthesis of leporin C (25), a cytotoxic hybrid PKS-NRPS natural product from Aspergillus, involves pericyclic steps catalysed by the stand-alone enzyme LepI.[70] Formation of a stable ketone intermediate by LepA, G and H is followed by reduction catalysed by LepF,ashort-chain dehydrogenase, generating a secondary alcohol. In the absence of LepI this
compound can spontaneously dehydrate, leading to the formation of quinone methide stereoisomers which then undergo Diels–Alder (DA) or hetero-Diels-Alder (HDA) cyclisations to form a mixture of adducts (Scheme 4C, red arrows). When LepI was included in the reaction, only two products were observed: the major product leporin C (25), the E-HDA-adduct, and lower quantities of the E-DA-adduct 26, which over time was converted into leporin C (25). Experimentally, LepI was observed to catalyse stereoselective dehydration to yield the Emethide intermediate 27, which is crucial for establishing the
ultimate product stereochemistry. The enzyme was found to promote formation of the HDA-adduct 25 compared to the DA-adduct 26 (1:1 with LepI, 94:6 without LepI). Furthermore, LepI can then catalyse a retro-Claisen rearrangement of 26, converting it into 25. This is the first reported enzyme catalysing a retro-Claisen rearrangement, and the reaction was catalysed with an impressive rate enhancement of 1.8V105(kcatversus knon).

In a similar manner to SpnF, LepI has sequence homology to O-methyl-transferases and utilises SAM as a co-factor, despite no methylation steps appearing in the leporin biosynthesis. Experiments with SAM, SAH and the charged mimic sinefungin established that the positively charged co-factor was important for both dehydration and retro-Claisen activities. Computational DFT analysis indicates that the initial (hetero)-Diels–Alder step is controlled by an ambimodal transition state, and the enzyme shifts the post-transition state bifurcation to favour direct production of the HDA adduct 25. Furthermore, the
analysis also supports the role of the SAM positive charge in lowering the energy of the ambimodal and retro-Claisen transition states.

A recently published structure of LepI in complex with SAM has provided insight into the mechanism of this multifunctional enzyme.[71] Computational docking of substrates indicate residues H133 and D296 are involved in the dehydration step, whilst the selectivity of the pericyclic reactions is a result of conformational restraints provided by hydrophobic residues.

The preferred pose of the docked intermediate 27 is more conducive to HDA rather than DA cyclisation, which supports the empirically measured enzyme selectivity. Further structural and mutational investigations of this fascinating enzyme will provide further insight into the role of SAM and how its various activities are coupled. This system is ideal for investigation of enzymatic ambimodal cycloaddition transition states since both products can be identified.

7. Putting the lid on an entropy trap
Coercing reaction partners into a conformation leading to a desired cycloaddition comes at a high entropic cost[5] which the enzyme should reduce for effective catalysis. How is this achieved? Catalytic champions among the natural [4+2] cyclases are the enzymes involved in tetronate and tetramate formation in the abyssomycin, versipelostatin and pyrridomycin biosynthesis.[13,72,73] The 4V104-fold rate acceleration observed with AbyU from abyssomycin synthesis,[73] comparable in magnitude to the Lewis acid catalysis with aluminum trichloride, and the transition state affinity of 3.9V108m@1(catalytic proficiency, [kcat/KM]/kuncat)[15] are excellent for a cyclase. Interestingly, most of the outstanding efficiency appears to be not owed to individual catalytic residues but to the cooperative action of multiple residues in a lid covering the active site.[74]

The structure of PyrI4 from pyrridomycin synthesis with product bound has illuminated details of the reaction mechanism (Figure 1 A).[74] Although Gln115 and His117 are close to the carbonyl group of the dienophile, they show little mutational sensitivity. In the Gln115Ala mutant, kcat only drops by a factor of three and remains unchanged in the His117Ala mutant. However, removal of an N-terminal helix covering the active site and mutation of individual salt bridge forming residues in this helix abolishes activity (Figure 1A). The N-terminal lid apparently holds the transition state tight in the active site but then quickly releases the product. Comparable lid-like movements have been predicted for AbyU, a homologue of PyrI4. Discrimination of the fine differences between transition state and product combined with a dynamic release mechanism might be key to efficient entropy trapping in [4+2] cyclases. Lid closure might force the substrate into the reactive conformation in an induced-fit like mechanism and thus reduce the entropy of activation.

8. Leading peptides to cyclisation
In the induced fit mechanism described above, substrate binding and catalysis are intimately coupled, but binding and catalysis can also occur at distinct locations in the enzyme structure. Ribosomal peptide natural products (RiPPs) are appended with cleavable leader peptides as universal binding handles to recruit various enzymes to work on the peptide backbone. Since the leader peptide guarantees binding, structural modifications are well tolerated in the core peptide, which has been exploited for combinatorial biosynthesis.[75] This enzymatic strategy, in which binding and catalysis are separated, seems to make RiPPs highly evolvable.

Pyridine synthases like TbtD have the unique ability to introduce pyridine residues into RiPPs via an aza-cycloaddition reaction.[76–79] In the case of the antibiotic thiomuracin, dehydration of two serine residues to dehydroalanine in the precursor peptide prepares for the pyridine synthase reaction catalysed by TbtD (Figure 1 B). The dienophile is the first residue of the core peptide and the diene partner is presumably generated by tautomerization of a downstream dehydroalanine to an iminol.

After cycloaddition, pyridine formation is completed by dehydration and elimination of the leader peptide. Structurally, three insertions make the difference between related, noncyclising dehydration elimination domains and the pyridine synthase TbtD.[76] One structure in complex with the leader peptide shows it to interact with the second insertion. Another structure in complex with truncated pyridine product 28 shows its binding at the third insertion (Figure 1 C). Ligand binding enhances the structural order in the third insertion, perhaps indicating an induced-fit like mechanism similar to the one observed with the aforementioned lid. The leader peptide binds to the enzyme with a low micromolar dissociation constant and its absence abolishes binding and reactivity.[76–79] In addition to the leader peptide, pyridine synthases require Cterminal recognition elements, possibly to distinguish between multiple potential dehydroalanine reaction partners.[78] Knowledge of the recognition elements enables the biocatalytic application of pyridine synthases on unnatural, possibly synthetic peptide substrates.[78,80] Still mostly in the dark, however, are catalytic details regarding the [4+2] cycloaddition.

9. Polar, bimolecular and stepwise
Intermolecular reactions render a synthesis convergent and thus break one large synthetic problem down into several small ones. One iconic example from total synthesis illustrating the utility of intermolecular, asymmetric Diels-Alder reactions is Liu and Jacobsen’s synthesis of the antifungal natural product ambruticin.[81] They employed Diels–Alder reactions under control of a chiral chromium catalyst to assemble two pyran rings at both ends of the molecule. Hence, intermolecular cycloaddition reactions are particularly valuable in the total synthesis for the build-up of complex scaffolds. Yet, almost all natural [4+2] cyclases known today have only been shown to promote intramolecular reactions. In nature, rare examples of intramolecular cycloaddition catalysts are riboflavin synthase and macrophomate synthase.

9.1. Riboflavin synthase
Riboflavin synthase catalyses an intermolecular cyclisation reaction that is part of primary metabolism and should therefore be old on an evolutionary time scale. This reaction leads to the biosynthesis of the essential cofactor riboflavin via pentacyclic intermediate 29 (Figure 2 A).[82,83] Several mechanisms have been proposed for the riboflavin synthase-catalysed dimerisation of 6,7-dimethyllumazine 30 but DFT calculations in water have ruled out most of them.[84] Among the energetically disfavoured routes were a radical tautomerization followed by stepwise cyclization and hydride transfer followed by Diels–Alder cyclization.[84,85] The mechanism is favoured on computational grounds, compatible with kinetic isotope effects[86] and involves a tautomerization of one reaction partner to diene-diamine 31 and sequential nucleophilic additions of the 6- and 7-methylene groups to the 7- and 6-carbon atoms of the other reaction partner.

The proposed riboflavin synthase mechanism is so appealing because only acid/base chemistry is required—a speciality of enzymes. Several transition states might be relevant for catalysis. Those leading to C@H deprotonation of the 6- and 7- methyl groups cause large deuterium isotope effects of 5 and 1.3-fold, respectively.[86] Computation predicts both carbon–carbon bond formation events to be energetically relevant too, with activation energies in the range of 20 kcalmol@1. Both barriers are lowered when the electrophilic reactant is protonated at one of the nitrogen atoms.

Calculations of the Gibbs energy profile were conducted in water, so participation of enzyme side chains could not be accurately predicted. However, an earlier crystal structure with substrate analogue bound[87] shows residues conserved among archaeal enzymes that might function as proton transfer catalysts (Figure 2 A). Asp73, for instance, is suggestively positioned to participate in the first C@C bond formation.[87] Proximity of residue Cys76 to the ligands raises the possibility of additional nucleophilic catalysis but this residue is not strictly conserved.[84,87]

9.2. Macrophomate synthase
Another example of an intermolecular cyclase is the fungal enzyme macrophomate synthase.[88–91] This enzyme catalyses a net-cycloaddition between enolpyruvate (32) and 2-pyrone 33(Figure 2 B). However, the cycloaddition product 34 is not directly detectable as it undergoes immediate decarboxylation to the known intermediate 35, which then dehydrates non-enzymatically to reveal macrophomate.[90] There are multiple indications that the initial enzyme-catalysed cyclisation occurs in a stepwise manner rather than via a concerted Diels-Alder mechanism. These include the enzyme’s phylogenetic classification as an aldolase, its proven aldolase side-reactivity[91] and QMMM calculations suggesting a Michael-aldol mechanism.[92]

The proposed stepwise macrophomate synthase mechanism involves the generation of a reactive enolate intermediate which undergoes a 1,6-Michael addition followed by an aldolring closure. The presence of an enolate intermediate and Michael addition reaction resembles the mechanism of iridoid synthase (Section 4.2), though in iridoid synthase the addition occurs at the 1,4-position.[34,41,43] Notably, macrophomate synthase also catalyses 1,2-additions of enolpyruvate toarange of aldehyde acceptors with potential synthetic applications.[91] In contrast to the reductive enolate formation in iridoid synthase, macrophomate synthase generates the Michael donor by Mg2+-assisted decarboxylation of oxaloacetate to enolpyruvate (32).

Participation of the enzyme in the cycloaddition reaction is evident from the enantioselectivity which is probably caused by blocking attack from one face of the enolpyruvate (Figure 2 B).[88] Furthermore, structural investigations led to the identification of an arginine residue (Arg101) that could stabilise the negative charge forming on the 2-pyrone during Michael addition. The “dienophile” pyruvate coordinated to the metal centre in a bidentate fashion with one face exposed to the diene resembles the arrangement in chiral catalysts and artificial metalloenzymes (Scheme 1). However, the metal here
serves as the electron sink for decarboxylation, not for narrowing the energy gap of a cycloaddition.

To regard [4+2] cyclases with a stepwise mechanism as“failed Diels–Alderases” is probably a mistake. Quantum-mechanical calculations of Diels–Alder reactions with various dienophiles show two trends:[93] a) more polar reactions involving more charge transfer proceed faster and b) more polar or even ionic Diels–Alder reactions tend to be less synchronous. There fore, rate acceleration in Diels–Alderases may be achieved through increasing the polar character of the reaction, which in the extreme would result in a stepwise reaction. By taking a highly polar route to the products via anionic intermediates, macrophomate synthase achieves an overall kcat of 0.6 s@1.[89]

10. Designer cyclases
Enzyme designers aim to meet the need of synthetic chemistry for fast and specific catalysts through the development of tailor-made enzymes. Novel enzymes catalysing cycloadditions have always been a prime target for these approaches. In contrast to current artificial metalloenzymes which embed a metal based cyclisation catalyst in a largely inert scaffold, de novo designs have active sites constructed from scratch—usually employing the standard repertoire of proteinogenic amino acids.

10.1. Catalytic antibodies
Catalytic antibodies offer important lessons about cycloaddition catalysis and cyclase design.[94] Before computers became powerful enough to predict transition-state and protein active site geometries, raising catalytic antibodies against transitionstate analogues was a promising strategy for designing enzyme catalysts for unnatural reactions. Despite some initial successes, catalytic antibodies have fallen out of favour because their catalytic efficiencies remained inferior to biological catalysts, possibly due to limitations of the antibody scaffold.[94]

The origins of cycloaddition catalysis have been investigated in detail with catalytic antibody 1E9 raised against hapten 36(Figure 3 A).[2] This designer catalyst shows a catalytic proficiency of 1.4V107m@1
,[94] well in the range of natural [4+2] cyclases. Surprisingly, 1E9 does not act as an entropy trap; whilst the enthalpy of activation determined from the temperature dependence of kcat is lowered by 4.2 kcalmol@1, the entropy of activation remains equally unfavourable for the catalysed and uncatalyzed reaction (@22.1 and @21.5 calmol@1K@1, respectively).[95] According to molecular dynamics simulations, residue Asn-H35 is crucial for the enthalpic effect as it hydrogen bonds to the carbonyl oxygen of the dienophile more strongly in the transition state than in the ground state. The catalytic interaction is bolstered by the hydrophobic environment with perfect shape complementarity to the transition state (Figure 3A). Product inhibition, a possible side-effect of strong transition state binding in Diels-Alderases, is avoided here through elimination of sulphur dioxide from product 37.

10.2. Computational design
For designer enzymes to become relevant for chemical synthesis, we must develop computational methods to design them in a short amount of time and with a high level of accuracy.[96] A computational cyclase design from the Baker lab, DA_20_00,[97] employs a glutamine and a tyrosine side chain interaction with the diene 38 and the dienophile N,Ndimethylacrylamide (39; Figure 3 B), but only reaches a catalytic proficiency of 8.7V103m@1. Subsequent optimization by cassette mutagenesis and error-prone PCR has yielded DA_20_20 with a 620-fold higher catalytic proficiency due to a 45-fold improved kcat and slightly improved KM values (3.5 and 3.9-fold for the diene and dienophile, respectively).[97,98] Installation of a computationally designed lid covering the active site and subsequent evolutionary optimization increased the catalytic proficiency to 8.4107m@1,[98,99] only five-fold below the natural bestin-class AbyU discussed above (Section 7). The resulting catalyst CE20 (Figure 3 B) displays the excellent stereoselectivity expected from a tightly tailored enzyme binding pocket. At present, the high demand for time and manpower make de novo enzyme design approaches impracticable for biocatalytic applications. However, the astonishing accuracy of the CE20 design and the growing reliability of the underlying in silico and in vitro optimisation protocols hold great promise for the future.

11. Discussion
11.1. Cyclases: a patchwork family
With a range of natural [4+2] cyclases now identified and some of them characterised in detail, the question can be asked: How do they evolve and why does this process appear so rare? With the exception of riboflavin synthase, which catalyses a reaction in primary metabolism, [4+2] cyclases seem to have appeared relatively late in evolution: the enzymes are phylogenetically highly diverse without any apparent sequence relation between the different classes, and each class occurs in a small group of organisms as part of a specialised/secondary metabolic pathway (Table 1). The cyclase classes appear to
have evolved independently to solve very specific metabolic questions, and consequently [4+2] cyclases have evolved multiple times from a wide range of unrelated enzymes, including redox enzymes, aldolases, and polyketide synthases.

One of the most proficient [4+2] cyclases, AbyU, is a small beta barrel with only 141 residues. If small and efficient [4+2] cyclases can exist, why did cycloaddition reactions not become more widespread early in evolution? Perhaps the limiting factor is not the evolution of the cyclase but rather the presence of suitable substrates. For a [4+2] cycloaddition to proceed, diene, dienophile and, usually, at least one conjugated functional group for electronic activation must preexist in a spatial arrangement permissive for reaction. A search in the Escherichia coli metabolome comprising 3760 metabolites, for instance, reveals only a few all-carbon dienes mostly related to aromatic amino acid metabolism and a few compounds which might be erroneously assigned to E. coli metabolism (retinal, leukotriene).[100]

There are limited opportunities for a novel[4+2] cyclase to emerge in this model organism and likely in many other species. Therefore, it seems that the rarity of cyclases is a consequence of the rarity of the requisite diene and dienophile systems.

11.2. Stabilizing or chaperoning the transition state
Fundamental, physico-chemical limitations of [4+2] cycloaddition catalysis could also hinder cyclase evolution. Both natural enzymes and designer catalysts tend to be sluggish catalysts in terms of rate acceleration (kcat/kuncat) and catalytic proficiency([kcat/KM]/kuncat; Table 2) compared to some enzymes from primary metabolism with proficiencies up to 2.0 1023m@1.[15] In cases where uncatalyzed cyclization rates are fast but undesired products are present, for example, in iridoid biosynthesis, the enzyme active site could mostly act as a chaperone for the transition state, similar to the dirigent proteins proposed to exert stereochemical influence in lignan biosynthesis.[101]

Rather than lowering the energy of the cycloaddition transition state, as one would expect a typical enzyme to do, these‘chaperone’ proteins may instead bias the reaction trajectory by other means. For example, they may destabilise undesired reaction trajectories, which in effect can lead to reaction selectivity without stabilisation of a transition state (i.e. negative catalysis). Other proteins may exert dynamic control of bifurcation following ambimodal transition states (e.g. SpnF, LepI) which can bias a reaction outcome without necessarily increasing reaction rate.

Two effects might explain the poor transition state binding of cyclases. First, the proximity effect for rather hydrophobic substrates is already high in water and cannot be fully exploited for enzyme catalysis.[22] Second, the similarity between the transition state and the products renders selective transitionstate stabilization difficult.[103] In particular, the potential for one of the most important enzyme functions, electrostatic preorganization,[104] might be low in reactions with hydrophobic partners. These limitations notwithstanding, a cyclase with a rate acceleration of 104 fold combined with enzyme-like selectivity can still be tremendously useful for biocatalysis.

12. Outlook
Exciting discoveries of natural [4+2] cyclases have encouraged a growing number of researchers to sift through biosynthetic pathways to find new members of this diverse family of enzymes. Many [4+2] cyclases are likely still awaiting discovery, but not every search will be rewarded with a new enzyme—some biological cyclisation reactions proceed without a catalyst.[105] Finding more bimolecular [4+2] cyclases, which are suspected in a number of pathways,[106] would be desirable in particular because of their potential synthetic utility. Cycloadditions are more widely used in chemical synthesis than in biosynthesis. Therefore, there is a demand for [4+2] cyclases that nature may not be able to fulfil. Enzyme engineers are working hard to expand the toolkit of organic chemists with efficient, tailor made cyclases. Great potential seems to reside in artificial metalloenzymes, which could possibly reach the efficiencies of typical, natural enzymes if they were subjected to extensive genetic optimization.[31] A growing number of natural and designed [4+2] cyclases will hopefully become available in the years to come and finally equip one of the most important reactions in synthetic chemistry with versatile
biocatalysts.

Acknowledgements
This work was kindly supported by grants of the European Research Council (311363), BBSRC (BB/J004561/1) to S.E.O and a Daimler und Benz fellowship (H.K.). B.R.L. acknowledges funding from UK Biotechnological and Biological Sciences Research Council (BBSRC) and Engineering and Physical Sciences Research Council (EPSRC) joint-funded OpenPlant Synthetic Biology Research Centre (BB/L014130/1). We thank Dr. L. Caputi for helpful discussions.

AA Blocks is committted to providing highly specialized chemicals，but more cheaper than sigma-aldrich:

Terephthalic acid Catalog No.:AA00002V CAS No.:100-21-0 MDL No.:MFCD00002558 MF:C8H6O4 MW:166.1308	1-(4-Nitrophenyl)ethanone Catalog No.:AA00002X CAS No.:100-19-6 MDL No.:MFCD00007355 MF:C8H7NO3 MW:165.1461
N,N-Dimethyl-4-nitroaniline Catalog No.:AA00002U CAS No.:100-23-2 MDL No.:MFCD00014712 MF:C8H10N2O2 MW:166.1772	1,4-Benzenedicarbonyl dichloride Catalog No.:AA00002W CAS No.:100-20-9 MDL No.:MFCD00000693 MF:C8H4Cl2O2 MW:203.0222
4-Nitroanisole Catalog No.:AA00002Z CAS No.:100-17-4 MDL No.:MFCD00007327 MF:C7H7NO3 MW:153.1354	1-Ethoxy-4-nitrobenzene Catalog No.:AA00003U CAS No.:100-29-8 MDL No.:MFCD00007330 MF:C8H9NO3 MW:167.1620
4-Cyanopyridine Catalog No.:AA00003F CAS No.:100-48-1 MDL No.:MFCD00006417 MF:C6H4N2 MW:104.1094	4-Vinylpyridine Catalog No.:AA00003K CAS No.:100-43-6 MDL No.:MFCD00006447 MF:C7H7N MW:105.1372
Benzyl bromide Catalog No.:AA00003O CAS No.:100-39-0 MDL No.:MFCD00000172 MF:C7H7Br MW:171.0345	Benzonitrile Catalog No.:AA00003G CAS No.:100-47-0 MDL No.:MFCD00001770 MF:C7H5N MW:103.1213
Ethylenzene Catalog No.:AA00003M CAS No.:100-41-4 MDL No.:MFCD00011647 MF:C8H10 MW:106.1650	Benzylamine Catalog No.:AA00003H CAS No.:100-46-9 MDL No.:MFCD00008106 MF:C7H9N MW:107.1531
3-Cyclohexene-1-carboxaldehyde Catalog No.:AA00003D CAS No.:100-50-5 MDL No.:MFCD00001572 MF:C7H10O MW:110.1537	Ethanethiol, 2-(diethylamino)- Catalog No.:AA00003P CAS No.:100-38-9 MDL No.:MFCD00066258 MF:C6H15NS MW:133.255
4,4'-(Pentamethylenedioxy)dibenzamidine Catalog No.:AA00003R CAS No.:100-33-4 MDL No.:MFCD00599574 MF:C19H24N4O2 MW:340.4195	4,4'-Dinitrodiphenyl disulfide Catalog No.:AA00003S CAS No.:100-32-3 MDL No.:MFCD00003573 MF:C12H8N2O4S2 MW:308.3329
4,4'-(Ethene-1,2-diyl)dibenzoic acid Catalog No.:AA00003T CAS No.:100-31-2 MDL No.:MFCD00013994 MF:C16H12O4 MW:268.2641	4-Nitrophenyl Isocyanate Catalog No.:AA00003V CAS No.:100-28-7 MDL No.:MFCD00007306 MF:C7H4N2O3 MW:164.1183
4-NITROPHENETHYL ALCOHOL Catalog No.:AA00003W CAS No.:100-27-6 MDL No.:MFCD00010202 MF:C8H9NO3 MW:167.1620	2,5-Pyridinedicarboxylic Acid Catalog No.:AA00003X CAS No.:100-26-5 MDL No.:MFCD00006297 MF:C7H5NO4 MW:167.1189
1-Azabicyclo[2.2.2]octane Catalog No.:AA00003Y CAS No.:100-76-5 MDL No.:MFCD00006690 MF:C7H13N MW:111.1848	1-Nitrosopiperidine Catalog No.:AA00003Z CAS No.:100-75-4 MDL No.:MFCD00023652 MF:C5H10N2O MW:114.1457
2H-Pyran-2-carboxaldehyde, 3,4-dihydro- Catalog No.:AA000041 CAS No.:100-73-2 MDL No.:MFCD00059170 MF:C6H8O2 MW:112.1265	N-Ethylmorpholine Catalog No.:AA000040 CAS No.:100-74-3 MDL No.:MFCD00006177 MF:C6H13NO MW:115.1735
2-Ethylpyridine Catalog No.:AA000043 CAS No.:100-71-0 MDL No.:MFCD00006361 MF:C7H9N MW:107.1531	2-ethenylpyridine Catalog No.:AA000045 CAS No.:100-69-6 MDL No.:MFCD00006355 MF:C7H7N MW:105.1372
2-Cyanopyridine Catalog No.:AA000044 CAS No.:100-70-9 MDL No.:MFCD00006218 MF:C6H4N2 MW:104.1094	(Tetrahydro-2H-pyran-2-yl)methanol Catalog No.:AA000042 CAS No.:100-72-1 MDL No.:MFCD00006624 MF:C6H12O2 MW:116.1583
Cyclohexanemethanol Catalog No.:AA00003E CAS No.:100-49-2 MDL No.:MFCD00001510 MF:C7H14O MW:114.1855	N-Methylaniline Catalog No.:AA00004D CAS No.:100-61-8 MDL No.:MFCD00008283 MF:C7H9N MW:107.1531
1-Phenyl-1-thiaethane Catalog No.:AA000046 CAS No.:100-68-5 MDL No.:MFCD00008559 MF:C7H8S MW:124.2034	N-Phenylhydroxylamine Catalog No.:AA000049 CAS No.:100-65-2 MDL No.:MFCD00045718 MF:C6H7NO MW:109.1259
3-Pyridinemethanol Catalog No.:AA00004J CAS No.:100-55-0 MDL No.:MFCD00006407 MF:C6H7NO MW:109.1259	potassium phenolate Catalog No.:AA000047 CAS No.:100-67-4 MDL No.:MFCD00036027 MF:C6H5KO MW:132.2016
Phenylmagnesium chloride Catalog No.:AA00004F CAS No.:100-59-4 MDL No.:MFCD00000464 MF:C6H5ClMg MW:136.8619	N-Methylcyclohexylamine Catalog No.:AA00004E CAS No.:100-60-7 MDL No.:MFCD00003832 MF:C7H15N MW:113.2007
Phenylhydrazine Catalog No.:AA00004B CAS No.:100-63-0 MDL No.:MFCD00007573 MF:C6H8N2 MW:108.1411	1,1,3,3,5,5,7,7-Octamethyltetrasiloxane Catalog No.:AA00004U CAS No.:1000-05-1 MDL No.:MFCD00039789 MF:C8H26O3Si4 MW:282.6322
N-Acetyl Sulfamethazine Catalog No.:AA00004Z CAS No.:100-90-3 MDL No.:MFCD00185750 MF:C14H16N4O3S MW:320.3668	Anisole Catalog No.:AA000048 CAS No.:100-66-3 MDL No.:MFCD00008354 MF:C7H8O MW:108.1378
Phenylmercuric Chloride Catalog No.:AA00004I CAS No.:100-56-1 MDL No.:MFCD00000463 MF:C6H5ClHg MW:313.1469	Cyclohexanone oxime Catalog No.:AA00004A CAS No.:100-64-1 MDL No.:MFCD00001660 MF:C6H11NO MW:113.1576
Phenylmagnesium bromide Catalog No.:AA00004G CAS No.:100-58-3 MDL No.:MFCD00000038 MF:C6H5BrMg MW:181.3129	Cyclohexylsulfamic acid Catalog No.:AA000050 CAS No.:100-88-9 MDL No.:MFCD00065234 MF:C6H13NO3S MW:179.2373
3-Cyanopyridine Catalog No.:AA00004K CAS No.:100-54-9 MDL No.:MFCD00006372 MF:C6H4N2 MW:104.1094	Benzyl mercaptan Catalog No.:AA00004L CAS No.:100-53-8 MDL No.:MFCD00004867 MF:C7H8S MW:124.2034
Benzenemethanesulfonic acid Catalog No.:AA000051 CAS No.:100-87-8 MDL No.:MFCD01657399 MF:C7H8O3S MW:172.2016	Benzenemethanaminium, N,N,N-trimethyl-, hydroxide (1:1) Catalog No.:AA000052 CAS No.:100-85-6 MDL No.:MFCD00008281 MF:C10H17NO MW:167.2481
3-Methylanisole Catalog No.:AA000053 CAS No.:100-84-5 MDL No.:MFCD00008395 MF:C8H10O MW:122.1644	3-Hydroxybenzaldehyde Catalog No.:AA000054 CAS No.:100-83-4 MDL No.:MFCD00003368 MF:C7H6O2 MW:122.1213
m-Tolylmethanamine Catalog No.:AA000056 CAS No.:100-81-2 MDL No.:MFCD00008118 MF:C8H11N MW:121.1796	3-Fluorobenzylamine Catalog No.:AA000055 CAS No.:100-82-3 MDL No.:MFCD00008113 MF:C7H8FN MW:125.1435
3-Methylstyrene Catalog No.:AA000057 CAS No.:100-80-1 MDL No.:MFCD00008617 MF:C9H10 MW:118.1757	1,2-Bis[(1,1-dimethylethyl)dimethylsilyl]hydrazine Catalog No.:AA00005E CAS No.:10000-20-1 MDL No.:MFCD07369154 MF:C12H32N2Si2 MW:260.5669
2-Chloro-3,5-dibromo-4-methylpyridine Catalog No.:AA000059 CAS No.:1000017-92-4 MDL No.:MFCD09864796 MF:C6H4Br2ClN MW:285.3637	N,N'-Methanediylidenebis(1,1,1-trimethylsilanamine) Catalog No.:AA00005M CAS No.:1000-70-0 MDL No.:MFCD00051538 MF:C7H20N2Si2 MW:188.4181
N-[[5-(3-Chlorophenyl)-3-hydroxy-2-pyridinyl]carbonyl]glycine Catalog No.:AA000061 CAS No.:1000025-07-9 MDL No.:MFCD30470233 MF:C14H11ClN2O4 MW:306.7011	1-Butyl tert-butyl ether Catalog No.:AA00005P CAS No.:1000-63-1 MDL No.:MFCD32206208 MF:C8H18O MW:130.2279
n-Butylchlorodimethylsilane Catalog No.:AA00005U CAS No.:1000-50-6 MDL No.:MFCD00015727 MF:C6H15ClSi MW:150.7218	Butyl Sodium Sulfate Catalog No.:AA00005O CAS No.:1000-67-5 MDL No.:MFCD00024891 MF:C4H9NaO4S MW:176.1666
2-Bromo-5-(diethoxymethyl)-3-methylthiophene Catalog No.:AA000065 CAS No.:1000018-59-6 MDL No.:MFCD06657808 MF:C10H15BrO2S MW:279.1939	6-Bromoimidazo[1,2-a]pyrazine-2-carboxylic acid Catalog No.:AA000067 CAS No.:1000018-56-3 MDL No.:MFCD09414724 MF:C7H4BrN3O2 MW:242.0296
methyl N-(2,2,4-trimethyl-3-oxo-1-phenyl pentyl)carbamate Catalog No.:AA000069 CAS No.:1000018-26-7 MDL No.:MFCD09864844 MF:C16H23NO3 MW:277.3587	4-Phenyl-1,3,5-triazine-2-thiol Catalog No.:AA000064 CAS No.:1000018-63-2 MDL No.:MFCD09702439 MF:C9H7N3S MW:189.2370
2-Chloro-3,5-dibromo-6-methylpyridine Catalog No.:AA000066 CAS No.:1000018-58-5 MDL No.:MFCD09702430 MF:C6H4Br2ClN MW:285.3637	3-Amino-2,6-dibromo-5-ethoxypyridine Catalog No.:AA00006G CAS No.:1000018-10-9 MDL No.:MFCD09864810 MF:C7H8Br2N2O MW:295.9592
ethyl 1-{[(tert-butoxycarbonyl)amino] sulfonyl}-4-piperidinecarboxylate Catalog No.:AA00006A CAS No.:1000018-25-6 MDL No.:MFCD09864838 MF:C13H24N2O6S MW:336.4045	Benzyl 4-(N-(tert-butoxycarbonyl)sulfamoyl)piperazine-1-carboxylate Catalog No.:AA00006E CAS No.:1000018-21-2 MDL No.:MFCD09864831 MF:C17H25N3O6S MW:399.4619
ethyl 2-[6-oxo-4-piperazino-1(6H)- pyridazinyl] acetate Catalog No.:AA00006B CAS No.:1000018-24-5 MDL No.:MFCD09864837 MF:C12H18N4O3 MW:266.2963	8-Bromo-6-chloroimidazo[1,2-a]pyridine-2-carboxylic acid Catalog No.:AA00006I CAS No.:1000017-98-0 MDL No.:MFCD22682816 MF:C8H4BrClN2O2 MW:275.4866
tert-butyl 4-[5-bromo-3-(methoxycarbonyl)-2- pyridinyl]tetrahydro-1(2H)-pyrazinecarboxylate Catalog No.:AA00006D CAS No.:1000018-22-3 MDL No.:MFCD09864832 MF:C16H22BrN3O4 MW:400.2676	ethyl 1-(6-oxo-1,6-dihydro-4-pyridazinyl)-4-piperidinecarboxylate Catalog No.:AA00006C CAS No.:1000018-23-4 MDL No.:MFCD09864836 MF:C12H17N3O3 MW:251.2817
5-Bromo-1-methyl-1H-Indazol-3-amine Catalog No.:AA00006H CAS No.:1000018-06-3 MDL No.:MFCD09864804 MF:C8H8BrN3 MW:226.0732	Ethyl 5-aminoimidazo[1,2-a]pyridine-2-carboxylate Catalog No.:AA00006J CAS No.:1000017-97-9 MDL No.:MFCD22989346 MF:C10H11N3O2 MW:205.2132
5-Chloroimidazo[1,2-a]pyridine-2-carboxylic acid Catalog No.:AA00006K CAS No.:1000017-93-5 MDL No.:MFCD09836168 MF:C8H5ClN2O2 MW:196.5905	(1-(tert-Butoxycarbonyl)-4-fluoro-1H-indol-2-yl)boronic acid Catalog No.:AA00006O CAS No.:1000068-25-6 MDL No.:MFCD11109421 MF:C13H15BFNO4 MW:279.0719
(1-(tert-butoxycarbonyl)-7-fluoro-1H-indol-2-yl)boronic acid Catalog No.:AA00006N CAS No.:1000068-65-4 MDL No.:MFCD11109422 MF:C13H15BFNO4 MW:279.0719	TERT-BUTYL (1-(4-AMINO-2-FLUOROPHENYL)PIPERIDIN-4-YL)CARBAMATE Catalog No.:AA00006T CAS No.:1000053-23-5 MDL No.:MFCD08443960 MF:C16H24FN3O2 MW:309.3791
2-Amino-4-(4-methoxyphenyl)-5-methylthiophene-3-carbonitrile Catalog No.:AA000075 CAS No.:100005-23-0 MDL No.:MFCD02609524 MF:C13H12N2OS MW:244.3122	Methyl 2-oxo-1,2,3,4-tetrahydroquinoline-7-carboxylate Catalog No.:AA00006U CAS No.:1000045-93-1 MDL No.:MFCD24387120 MF:C11H11NO3 MW:205.2099
2-(3-methylphenyl)isonicotinic acid Catalog No.:AA000079 CAS No.:100004-94-2 MDL No.:MFCD14666477 MF:C13H11NO2 MW:213.2319	5-Nitro-4-(trifluoromethyl)pyridin-2-amine Catalog No.:AA00007H CAS No.:1000152-83-9 MDL No.:MFCD28155019 MF:C6H4F3N3O2 MW:207.1101
tert-butyl 4-(2-chloro-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ylamino)piperidine-1-carboxylate Catalog No.:AA000081 CAS No.:1000207-52-2 MDL No.:MFCD28053709 MF:C17H25ClN4O2 MW:352.8590	3-(4-(Trifluoromethyl)phenyl)pyrrolidine Catalog No.:AA000083 CAS No.:1000198-76-4 MDL No.:MFCD12407011 MF:C11H12F3N MW:215.2149
2,6-Dibromo-4-(trifluoromethyl)pyridine Catalog No.:AA00007G CAS No.:1000152-84-0 MDL No.:MFCD22682722 MF:C6H2Br2F3N MW:304.8900	1-(tert-Butoxycarbonyl)-4,4-difluoropyrrolidine-2-carboxylic acid Catalog No.:AA00008R CAS No.:1000313-01-8 MDL No.:MFCD22121409 MF:C10H15F2NO4 MW:251.2272
2-(Pyridin-2-ylthio)acetic acid Catalog No.:AA00008O CAS No.:10002-29-6 MDL No.:MFCD00276114 MF:C7H7NO2S MW:169.2010	(1S,2S,5R,6S,7S)-6,8,8-Trimethyl-3-azatricyclo[5.1.1.02,5]nonan-4-one Catalog No.:AA00008S CAS No.:1000304-40-4 MDL No.:MFCD11505102 MF:C11H17NO MW:179.2588
(1R,2R,5S,6R,7R)-6,8,8-Trimethyl-3-azatricyclo[5.1.1.02,5]nonan-4-one Catalog No.:AA00008U CAS No.:1000304-34-6 MDL No.:MFCD11505101 MF:C11H17NO MW:179.2588	4-[5-(4-Chlorophenyl)-2-methyl-3-(1-oxopropyl)-1H-pyrrol-1-yl]benzenesulfonamide Catalog No.:AA00008Z CAS No.:1000279-69-5 MDL No.:MFCD16495814 MF:C20H19ClN2O3S MW:402.8945
3-(2-Bromophenyl)-5-(3-nitrophenyl)-1,2,4-oxadiazole Catalog No.:AA00009T CAS No.:1000339-27-4 MDL No.:MFCD09878377 MF:C14H8BrN3O3 MW:346.1356	3-(2-Bromophenyl)-5-(2-fluorophenyl)-1,2,4-oxadiazole Catalog No.:AA00009V CAS No.:1000339-25-2 MDL No.:MFCD09878375 MF:C14H8BrFN2O MW:319.1285
(4-Bromo-2-nitrophenyl)hydrazine hydrochloride Catalog No.:AA0000A3 CAS No.:100032-79-9 MDL No.:MFCD09260482 MF:C6H7BrClN3O2 MW:268.4957	2-Amino-5-bromoisonicotinic acid Catalog No.:AA00009X CAS No.:1000339-23-0 MDL No.:MFCD09878357 MF:C6H5BrN2O2 MW:217.0201
2-Fluoro-3-hydroxybenzonitrile Catalog No.:AA00009W CAS No.:1000339-24-1 MDL No.:MFCD09839222 MF:C7H4FNO MW:137.1112	2-Phenylpyrrolidine Catalog No.:AA0002D2 CAS No.:1006-64-0 MDL No.:MFCD01631835 MF:C10H13N MW:147.2169
Methyl 1H-indole-5-carboxylate Catalog No.:AA0003OP CAS No.:1011-65-0 MDL No.:MFCD00153023 MF:C10H9NO2 MW:175.1840	4-Acetamidophenylboronic acid Catalog No.:AA0003YQ CAS No.:101251-09-6 MDL No.:MFCD02179451 MF:C8H10BNO3 MW:178.9809
N-Boc-(methylamino)acetaldehyde Catalog No.:AA000KEY CAS No.:123387-72-4 MDL No.:MFCD08064267 MF:C8H15NO3 MW:173.2096	4-Bromo-2,6-difluorobenzonitrile Catalog No.:AA000L2Z CAS No.:123843-67-4 MDL No.:MFCD01310981 MF:C7H2BrF2N MW:217.9983
2-Iodo-5-methylaniline Catalog No.:AA000ZVN CAS No.:13194-69-9 MDL No.:MFCD00833395 MF:C7H8IN MW:233.0496	3,4,5-Trifluorobenzaldehyde Catalog No.:AA00106X CAS No.:132123-54-7 MDL No.:MFCD00083523 MF:C7H3F3O MW:160.0933
5-Bromobenzo[d]oxazole Catalog No.:AA0010D3 CAS No.:132244-31-6 MDL No.:MFCD03095026 MF:C7H4BrNO MW:198.0168	2,5-Dibromoterephthalic Acid Catalog No.:AA00130S CAS No.:13731-82-3 MDL No.:MFCD00204593 MF:C8H4Br2O4 MW:323.9230
2-Naphthoic acid Catalog No.:AA0033BT CAS No.:93-09-4 MDL No.:MFCD00004101 MF:C11H8O2 MW:172.1800	5-Bromoindole-3-carboxaldehyde Catalog No.:AA00345K CAS No.:877-03-2 MDL No.:MFCD00152016 MF:C9H6BrNO MW:224.0540
trans-4-Boc-aminocyclohexanol Catalog No.:AA0034HF CAS No.:111300-06-2 MDL No.:MFCD03844613 MF:C11H21NO3 MW:215.2893	Cyclopentadienyl(formylcyclopentadienyl)iron Catalog No.:AA0034UQ CAS No.:12093-10-6 MDL No.:MFCD00001429 MF:C11H20FeO MW:224.1209
tert-Butyl 6-oxo-2-azaspiro[3.3]heptane-2-carboxylate Catalog No.:AA00359Q CAS No.:1181816-12-5 MDL No.:MFCD15071430 MF:C11H17NO3 MW:211.2576	3,3-Diethoxypropionic acid ethyl ester Catalog No.:AA0035YE CAS No.:10601-80-6 MDL No.:MFCD00009865 MF:C9H18O4 MW:190.2368
2-(4-Aminophenyl)ethanol Catalog No.:AA0037XU CAS No.:104-10-9 MDL No.:MFCD00007922 MF:C8H11NO MW:137.1790	4-Bromophthalic Anhydride Catalog No.:AA0038B0 CAS No.:86-90-8 MDL No.:MFCD00191323 MF:C8H3BrO3 MW:227.0116
2-Bromoisonicotinonitrile Catalog No.:AA003GL9 CAS No.:10386-27-3 MDL No.:MFCD07367879 MF:C6H3BrN2 MW:183.0054	2-Methyl-1-Phenyl-2-Propanol Catalog No.:AA003HIR CAS No.:100-86-7 MDL No.:MFCD00004465 MF:C10H14O MW:150.2176
4-Biphenylcarboxylic acid Catalog No.:AA003KRE CAS No.:92-92-2 MDL No.:MFCD00002553 MF:C13H10O2 MW:198.2173	(4-Bromophenyl)methanol Catalog No.:AA003KWX CAS No.:873-75-6 MDL No.:MFCD00004650 MF:C7H7BrO MW:187.0339
6-Bromo-2-chloroquinazoline Catalog No.:AA003N10 CAS No.:882672-05-1 MDL No.:MFCD09261000 MF:C8H4BrClN2 MW:243.4878	Benzo[d][1,3]dioxol-5-amine Catalog No.:AA003NZ1 CAS No.:14268-66-7 MDL No.:MFCD00005832 MF:C7H7NO2 MW:137.1360
Boc-3-aminobenzoic acid Catalog No.:AA003OCL CAS No.:111331-82-9 MDL No.:MFCD00235884 MF:C12H15NO4 MW:237.2518	Diethylaminoethanol Catalog No.:AA003PEY CAS No.:100-37-8 MDL No.:MFCD00002850 MF:C6H15NO MW:117.1894
N,N,N,N-Tetramethyl-1,4-phenylenediamine Catalog No.:AA003SDV CAS No.:100-22-1 MDL No.:MFCD00008309 MF:C10H16N2 MW:164.2474	3-Bromo-2-chlorophenol Catalog No.:AA004O9F CAS No.:863870-87-5 MDL No.:MFCD08166321 MF:C6H4BrClO MW:207.4524
3,4-Dibromo-1H-pyrrole-2,5-dione Catalog No.:AA007TZD CAS No.:1122-10-7 MDL No.:MFCD00185696 MF:C4HBr2NO2 MW:254.8642	2,2-Dimethyl-1,3-dioxolane-4-methanol Catalog No.:AA007X71 CAS No.:100-79-8 MDL No.:MFCD00063238 MF:C6H12O3 MW:132.1577
N,N-Diethylethylenediamine Catalog No.:AA007XWL CAS No.:100-36-7 MDL No.:MFCD00008176 MF:C6H16N2 MW:116.2046	Fmoc-Ser(tBu)-Ser(psi(Me,Me)pro)-OH Catalog No.:AA008T1U CAS No.:1000164-43-1 MDL No.:MFCD11974988 MF:C28H34N2O7 MW:510.5788
(1-(tert-Butoxycarbonyl)-6-fluoro-2-indolyl)boronic acid Catalog No.:AA008T8A CAS No.:1000068-26-7 MDL No.:MFCD09953519 MF:C13H15BFNO4 MW:279.0719	2-(Benzo[b]thiophen-7-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane Catalog No.:AA008TW2 CAS No.:1000160-74-6 MDL No.:MFCD16995819 MF:C14H17BO2S MW:260.1596
(1-Methyl-1H-pyrazol-4-yl)methanol Catalog No.:AA008U5T CAS No.:112029-98-8 MDL No.:MFCD01822311 MF:C5H8N2O MW:112.1298	5(6)-Dehydro-4(5)-dihydro D-(-)-Norgestrel (>90%) Catalog No.:AA008UHJ CAS No.:100021-05-4 MDL No.:MFCD00797840 MF:C21H28O2 MW:312.4458
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophene Catalog No.:AA008UO0 CAS No.:1000160-75-7 MDL No.:MFCD13619876 MF:C14H17BO2S MW:260.1596	Fructosylvaline Catalog No.:AA008WEC CAS No.:10003-64-2 MDL No.:MFCD19443381 MF:C11H21NO7 MW:279.2869
3’-O-Desmethyl Etoposide Catalog No.:AA008WZH CAS No.:100007-54-3 MDL No.: MF:C28H30O13 MW:574.5300	α-Etoposide Catalog No.:AA008X3M CAS No.:100007-53-2 MDL No.:MFCD11109422 MF:C29H32O13 MW:588.5566
Etoposide Glucuronide Catalog No.:AA008YJD CAS No.:100007-55-4 MDL No.:MFCD15144997 MF:C35H40O19 MW:764.6807	3-(4-(Methylsulfonyl)phenoxy)benzoic acid Catalog No.:AA0090JB CAS No.:1000018-30-3 MDL No.:MFCD09264550 MF:C14H12O5S MW:292.3071
cis-Etoposide Catalog No.:AA0091UI CAS No.:100007-56-5 MDL No.:MFCD11109422 MF:C29H32O13 MW:588.5566	4,5,6,7-Tetrahydroisoxazolo[4,3-c]pyridine Catalog No.:AA0094P5 CAS No.:1000303-67-2 MDL No.:MFCD12405584 MF:C6H8N2O MW:124.1405
Benzenamine, 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)- Catalog No.:AA009524 CAS No.:1000339-10-5 MDL No.:MFCD16996341 MF:C13H20BNO3 MW:249.1138	4-[(4-Bromo-2-Fluorophenyl)Sulfonyl]Morpholine Catalog No.:AA0095H4 CAS No.:1000068-42-7 MDL No.:MFCD27392030 MF:C10H11BrFNO3S MW:324.1666
Methyl (2-hydroxybenzoyl)imidoformate Catalog No.:AA0099Z2 CAS No.:1000018-60-9 MDL No.:MFCD09998770 MF:C9H9NO3 MW:179.1727	7-(chloromethyl)-3-methyl-5H-[1,3]thiazolo[3,2-a]pyrimidin-5-one Catalog No.:AA009MFY CAS No.:100003-81-4 MDL No.:MFCD06655025 MF:C8H7ClN2OS MW:214.6720
3-(m-Tolyl)isonicotinic acid Catalog No.:AA00H91A CAS No.:100004-79-3 MDL No.:MFCD18207559 MF:C13H11NO2 MW:213.2319	3-chloro-2-fluoro-5-nitrobenzoic acid Catalog No.:AA00H91G CAS No.:1000162-34-4 MDL No.:MFCD23701946 MF:C7H3ClFNO4 MW:219.5544
Hexamethylenetetramine Catalog No.:AA00H913 CAS No.:100-97-0 MDL No.:MFCD23699472 MF:C6H12N4 MW:140.1863	D-Cysteine Catalog No.:AA00IGY0 CAS No.:921-01-7 MDL No.:MFCD00066461 MF:C3H7NO2S MW:121.1582
benzyl 4-[5-chloro-1-(2-ethoxy-2-oxoethyl) -6-oxo-1,6-dihydro-4-pyridazinyl]tetrahydro-1(2H)- pyrazinecarboxylate Catalog No.:AA00ISQ4 CAS No.:1000018-18-7 MDL No.:MFCD09864828 MF:C20H23ClN4O5 MW:434.8734	2-(4-(4-((Benzyloxy)carbonyl)piperazin-1-yl)-5-chloro-6-oxopyridazin-1(6H)-yl)acetic acid Catalog No.:AA00IWVE CAS No.:1000018-20-1 MDL No.:MFCD09864830 MF:C18H19ClN4O5 MW:406.8203
2-(2,6-dioxopiperidin-1-yl)ethane-1-sulfonyl chloride Catalog No.:AA0166S6 CAS No.:1000339-13-8 MDL No.:MFCD09864173 MF:C7H10ClNO4S MW:239.6766	3-(6,8-Dimethyl-[1,2,4]triazolo[4,3-b]pyridazin-7-yl)propanoic acid Catalog No.:AA018N7Y CAS No.:1000339-20-7 MDL No.:MFCD09881042 MF:C10H12N4O2 MW:220.2279
1-tert-Butyl 2-methyl 4,4-difluoropyrrolidine-1,2-dicarboxylate Catalog No.:AA01ABJ7 CAS No.:1000313-00-7 MDL No.:MFCD09263325 MF:C11H17F2NO4 MW:265.2538	Methyl 4-(5-aminopyridin-2-yl)piperazine-1-carboxylate Catalog No.:AA01AMO6 CAS No.:1000334-84-8 MDL No.:MFCD12413700 MF:C11H16N4O2 MW:236.2703
Anhydroheliotridine Catalog No.:AA01CBRL CAS No.:100009-90-3 MDL No.: MF:C8H11NO MW:137.1790	Ampicillin Amino-benzeneacetaldehyde Catalog No.:AA01DZ7K CAS No.:10001-82-8 MDL No.:MFCD11109422 MF:C24H26N4O5S MW:482.5520
2-Methylthioadenosine triphosphate (sodium salt) Catalog No.:AA01ENOR CAS No.:100020-57-3 MDL No.:MFCD08703597 MF:C11H14N5Na4O13P3S MW:641.1999	3-{[(benzyloxy)carbonyl]amino}adamantane-1-carboxylic acid Catalog No.:AA01FFSD CAS No.:10002-15-0 MDL No.:MFCD31451483 MF:C19H23NO4 MW:329.3902
DL-tryptophan ethyl ester hydrochloride Catalog No.:AA00004V CAS No.:1000-00-6 MDL No.:MFCD00067553 MF:C13H17ClN2O2 MW:268.7393	3-(2-Bromophenyl)-5-(2,4-dichlorophenyl)-1,2,4-oxadiazole Catalog No.:AA00009U CAS No.:1000339-26-3 MDL No.:MFCD09878376 MF:C14H7BrCl2N2O MW:370.0282
Triisobutylaluminum Catalog No.:AA00004W CAS No.:100-99-2 MDL No.:MFCD00008929 MF:C12H27Al MW:198.3243	Styrene Catalog No.:AA00003L CAS No.:100-42-5 MDL No.:MFCD00008612 MF:C8H8 MW:104.1491
Benzyl Alcohol Catalog No.:AA00003C CAS No.:100-51-6 MDL No.:MFCD00004599 MF:C7H8O MW:108.1378	1,4-Dinitrobenzene Catalog No.:AA00002T CAS No.:100-25-4 MDL No.:MFCD00007314 MF:C6H4N2O4 MW:168.1070