The Enigma of Sponge-Derived Terpenoid Isothiocyanate-Thiocyanate Pairs. A Proposal for their Biosynthesis

1. Introduction

Exotic terpenoid isonitriles (TIs, Figure 1) which occur exclusively within the domain of certain genera of marine sponges (Porifera), e.g. Acanthella, Adocia, Axinella, Axynissa, Cymbastella among others, and the seaslugs (nudibranchs) that prey upon them, have been known since the early 1970s. Marine isonitriles and their derivatives have been extensively reviewed [1,2,3]. Examples of cyclized terpenoid isonitriles and their accompanying α-adducts (1a-c, 2a–l, 3) are depicted in Figure 2. Only recently have certain members (e.g. 7,20-diisocyanoadociane (3) [4], kahilinols A (1a) [5] and B (1b) [6]) been identified and investigated as potential antimalarial therapeutics. Isonitrile 1b exhibits potent activity against the chloroquine-resistant plasmodium, Dd2 (IC₅₀ = 4.6 nM). MED6-189 (2), a synthetic analog developed from 1b, shows promising in vitro antiplasmodial activity (Dd2 IC₅₀ = 47± 7 nM) and strong efficacy in plasmodium-infected mice with no apparent toxicity or hemolytic liabilities [7].

TIs are often accompanied by the corresponding adducts generated by α-addition to the triple -NC bond; amides, carbonimidic dichlorides, and, most commonly, alkyl isothiocyanates [8]. Sponge-derived isonitrile biosynthesis can be rationalized by capture of late-stage terpenoid carbocations arising from C-C cyclizations and Wagner-Meerwein rearrangements of their activated precursors (GPP, FPP and GGPP) and final capture by a biosynthetic ‘NC vector’ [9] to generate isonitriles, R-NC. Isothiocyanates, R-NCS, e.g. kalihinol M 1d) (Figure 2) and 2a follow as the products of sulfur-transfer reaction of low-valent S donors (e.g. glutathione dimer, thiosulfate, etc.) to R-NC. Terpenoid carbon backbones of TIs and related derivatives, including isothiocyanates (ITC, Figure 1) are mostly familiar in plant terpenoids, but notable exceptions are synthesized only by sponges. For example, the common plant sesquiterpene (C₁₅) skeletons of cadinane, drimane, and bisabolane are all represented in the sponge TI, ITC and TC family, but sponges and their predators (nudibranchs) are the exclusive provenance of axinyssane [10,11], pupukeanane [12,13], neopupukeanane and the diterpenes (C₂₀) adociane, [4], amphilectane [14,15], isoneoamphilectane [16] among others.

The startling discovery of co-localized thiocyanates (R-SCN), e.g. 4-thiocyanato-9-cadinene 2a [17], 2b [18] (with the same carbon skeleton as isonitrile 2j [12]), 2c-d [19], with carbon skeletons similar to those of the more common isothiocyanates (R-NCS), 2g,h [20], and 2m [21], presents an enigma [22]: ‘how can a linear biosynthetic pathway of R-NC –> R–NCS accommodate R–SCN?’ While the biosynthesis of allyl thiocyanate, the pungent volatile natural product found in mustard oil, horseradish, garlic and many other cruciferous plants and its isomerized regioisomer, allyl isothiocyanate, has long-been understood (decomposition of the glucosinolate, sinigrin, derived from Met [23]), the origin of sponge-derived thiocyanates is puzzling. Now, this enigma is addressed by consideration of a putative interplay of three integrated biosynthetic sequelae: capture of the terpenoid carbocation R⁺ by cyanoformate, NC-COOH (i) [9] (itself in equilibrium with NC^– and CO₂ and derived from Gly), reactions promoted by co-localized rhodanese – a dual-function enzyme that can both convert inorganic NC^– to thiocyanate ion, NCS^–, and alkyl isonitriles to alkyl isothiocyanate (R-NC –> R–NCS) – and promiscuous terminal interception of terpenoid carbocations by co-liberated inorganic NCS^–.

2. Results and Discussion.

2.1. Chemistry and Bonding in TC and ITC.

While hundreds of marine-derived TIs, ITCs and FAs have been described., only six TCs are known. The structures of the TCs were examined on a case-by-case basis, with their isomeric or similar isothiocyanates (ITC). ‘Reverse engineering’ of the likely terminal steps in their biosynthesis by probing the thermodynamics of bond-breaking and bond-forming provides insights into what could be possible and what is improbable. For the purposes of informed discussion, a brief review of the chemistry of the cumulated-bond functional group, NCS, in TC and ITC molecules is in order. TC and ITC isomers are easily differentiated spectroscopically by ¹³C NMR and IR. ITC shows a very intense, broad stretching frequency at ~2100 cm^–1, while the TC IR band is of similar frequency, but narrow and of medium intensity. The ¹³C NMR signal of C at the point of attachment, reflects the difference in electronegativity of S and N: ITC shows C-N_α δ ~ 75 while the C-S_α signal in TC is δ ~ 64 ppm [22].

The isomers t-butylthiocyanate (4) and t-butylisothiocyanate (5) (Figure 3) serve as convenient models for comparisons to their terpenoid natural product counterparts. Bonding in the triatomic grouping NCS differs between TC and ITC. DFT calculations of the optimized geometries and energies of each (Figure 1) shows, as expected, the geometry of N-C-S is linear due to sp hybridization at C (ω = 179.8 ˚ and ω = 179.2˚, respectively), isoelectronic with alkyl azides, consistent with X-ray crystal structures of natural product ITCs [24] and TCs [17]. The bondings of the heteroatom attached to the alkyl group C (notated here as the α atom) differ significantly. The corresponding bond angles and bond lengths of 4 and 5 (θ(C-S-C) = 100˚ and (θ(C-N_α- C) = 176.8˚) reflect predominantly sp³ and sp hybridization, respectively, of the α-heteroatom attached to the t-Bu group. The shorter C-N_α bond length in 5 (d₂ = 1.44 Å is evident of a stronger σ bond between the t-Bu group and its α-heteroatom compared to 4 (d₁ = 1.88 Å), as expected for row 2 versus a row 3 elements, and recapitulates the thermodynamic stability of 5 over 4 [25].

ITCs are electrophilic: like isonitriles they undergo addition reactions at the cumulated bond, even with weak nucleophiles. TCs reluctantly undergo nucleophilic substitution reactions at the attached C when it 1˚ or 2˚, but not 3˚. Both allylic ITCs and TCs may participate in [3,3]-sigmatropic rearrangement (see below).

Three scenarios that may account for generation of TC natural products are considered.

2.2. Dissociation-Reassociation.

The isomerization of TC to ITC was examined as a possible linear pathway linking the corresponding TI (R-NC –> R–NCS –> R-SCN). Of necessity, such an isomerization must follow a unimolecular rate law, R = k[R-NCS] and an S_N1 mechanism due to the 3˚ alkyl group that is almost invariably the point of heteroatom attachment in TC and TCI natural products. The leaving group ability of the thiocyano group in R-SCN is at best weak, while in R-NCS, it is considerably poorer due to the stronger R-N bond. For various reasons, the bond dissociation energy in each of the two isomers is not readily calculated, but we can obtain a qualitiative picture from the relative the rates of sigmatropic [3,3] rearrangements of isomeric allylic thiocyanate (e.g. 6) to isothiocyanates (e.g. 7) and the isoelectronic allyl azides (e.g. 8, Figure 4). The equilibrium constants, K_eq, of the reactions quantitatively reflect their relative stabilities. While the reaction rates of [3,3] rearrangement of allylic azides (Winstein rearrangement [26]) are fast (most rapidly isomerize at ambient temperatures [27]), CH₂=CH-NCS and CH₂=CH-SCN interchange more slowly [28]. For example, the isomerization of the latter to the former occurs upon distillation (b.p. 150-2 ˚C) [29]) and is favored for reasons of greater bond-strength in the product (vide supra) [30]. The likely explanation of the slower rate reaction of [3,3]-rearrangement of ITC-TC pairs is poor overlap of row 2 versus row 3 frontier orbitals in the transition state.

2.3. Precedence Strengthens the Role of Adventitious NCS^–

ITCs are expressed by bacteria from isonitriles by task-specific adapted rhodanese. Rhodanese is distributed widely in the Nature and carries out the important role of scavenging inorganic cyanide formed adventitiously in metabolism, e.g. ‘leakage’ from C-1 tetrahydrofolate metabolism or other cyanide-generating reactions [31,32,33]. A key intermediate, cyanoformate, NC-COOH, CF, is ephemeral in the biosynthesis of the plant hormone ethylene, CH₂=CH₂ from 1-aminocyclopropane-1-carboxylate [34]. In Burkholderia gladioli that produces the non-terpenoid isonitrile, Hertwick and coworkers showed that rhodanese RhDE, in addition to donating competency in scavenging cyanide NC^– into thiocyanate, NCS^–, catalyzes the substrate-specific sulfur transfer reaction R-NC –> R-NCS generating the ITC, sinapigladioside [35], by utilizing thiosulfate as an S donor [36]. Hertwick speculates that the enzyme was recruited from a, “ubiquitous detoxification enzyme for the formation of a bioactive specialized metabolite” [36]. The concept of recruitment of rhodanese, with evolved substrate-specificity to the latter task, may also find support in the biosynthesis of terpenoid ITCs in sponges, and the observation that TIs are not uniformly accompanied by their ITC counterparts.

2.4. Thiocyanate is a competent ambident nucleophile.

Pearson and coworkers compiled relative rates of nucleophilic substitutions of methyl iodide, CH₃I, with selected nucleophiles under comparable conditions (25 ˚C) [37]. The second-order rate constant, k₂, for reaction with NCS^– and NC^– (25 ˚C, Table 1) are similar (Entries 8 and 9: 5.74 x10^–4 and 6.5 x 10^–4 mol^–1.sec^-1, respectively (for comparison, k₂ for the reactions with thiophenoxide PhS^–, thiosulfate, S₂O₃^2– are 1.07 and 0.114 mol^–1.sec^-1 [37]).

A simplified scenario can be proposed for the terminal step in TC and ITC biosynthesis (Figure 5). S_N1 capture of NCS– at S (the more nucleophilic end) gives TC (path a). ITC is formed by S_N1 capture at N which contributes to the pool of ITC generated by S addition to the corresponding isonitrile, TI. Experimentally, it would be difficult to estimate the fraction of ITC that arises from paths b and c. As mentioned earlier, the results of radiolabeling experiments by NCS^– are somewhat equivocal and at this point do not lend insight into the contributions of paths b and c.

Inspection of the structures of secondary metabolites containing the R-SCN group (TCs) excludes the pre-described enzyme system for tailoring of R-NC to R-CNS, but still supports a role of rhodanese as a generator of the ambident nucleophile NCS^–. TCs may arise from adventitious capture of thiocyanate at S by S_N1 addition, a reaction that would also add to the pool of ITC from the former mechanism (Scheme 1).

The well-known rule of thumb for ambident nucleophilicity holds that the bond-forming reaction occurs at the less negative atom, e.g. enolate reactions with most electrophiles occur at the α-C center instead of O. The rule predicts TCs are kinetically favored over ITCs when NCS^– reacts with electrophiles, R-X (X= halogen) at S, but typically mixtures of TCs and ITCs are formed. It should be remembered that an extrapolation of the same reaction, but with replacement of NCS^–with NC^– gives only nitriles, R-CN, not isonitriles, by exclusive nucleophilic addition at C [9]. While HCN is a weak Brønsted acid (pKa = 9.25), HSCN is strong (pKa = –0.7 [38]). HSCN is fully ionized at physiological pH. In short, NCS^– is more an ‘equal opportunity’ nucleophile than NC^– . At equilibrium, isomerization would favor ITC over TC.

2.5. A Simple Proposal – Thiocyanates Arise Adventitiously.

How do these foregoing data affect interpretation of the provenance and distribution of alkyl isothiocyanate, ITC, versus alkyl thiocyanate, TC? Alkyl TCs are rarer than their isomeric ITCs. Biosynthetically, ITCs mostly arise from a linear pathway of TI –> ITC by addition of low-valent sulfur, the less abundant TCs more likely arise from adventitious nucleophilic capture of free thiocyanate, NCS^– (delivered by rhodanese) by terpenoid carbocations in a parallel pathway (Scheme 1). A convincing appreciation of this proposition is gained by analysis of reported isolation yields of TCs relative to their analogous conspecific ITCs from the same organism. Like the substitution products of NCS^– with electrophilic carbocationic precursors, ITCs are found to predominate over TCs. One remaining question is how can exogenous NCS^– assimilated by sponges that make TC-ITC pairs? While exogenous cyanide can intercept the intracellular dynamic equilibrium of cyanoformate (CF, NC-COOH) by passive diffusion in its neutral form, HCN, the pK_a of HSCN would seem prohibitively low (–0.7) for a similar process; a ‘thiocyanate ion transporter’ would need to be invoked [39].Thiohalobacter sp., with induced capacity to metabolize NCS^–, have been raised by repeated passage in NCS^– containing culture medium [40].

In each case where analogous TC and ITC pairs do co-occur, the structures of the terpenoid carbon skeletons differ; 2g,h and 2i are the exceptions [20]. An interpretation of this phenomenon is that the last step of S_N1 capture to form TI or TC is rate-dependent upon the structure of the incipient carbocation, R⁺ and determined by its collapse and capture of a NC delivery ‘vector’, as recently proposed (Figure 4, [9]) or, in the latter case, free NCS^–. If R⁺ is more persistent (longer half-life), the S_N1 capture by ‘off-pathway’ NCS^– becomes more competitive, leading to product distributions favorable to TC.

Natural product allylic thiocyanates, e.g. farnesyl isothiocyanate and thiocyanate (8b,c, Figure 1), hypothetically obtainable from nucleophilic substitution of farnesyl pyrophosphate, or 8b separately by S-transfer to farnesyl isonitrile (8a, which itself, “remains elusive” [1]), are kinetically labile. TCs in time would be expected isomerize to their more substituted tert-allylic NCS counterparts (e.g. 9). The majority of sponge-derived TCs, however, are ‘fixed’; stabilized by substitution at 3˚ alkyl groups. While farnesyl isothiocyanate (8b) [41] and formamide, 8d, are known [42], but the foregoing reasons make it unlikely the ‘missing’ 8c (or 8a?) will ever be isolated from natural sources as [3,3]-rearrangement irreversibly converts 8c to its isomeric nerolidyl isothiocyanate isomer, 9. The fact that 9 has not been found in Nature suggests that 8c, too, is absent. From the single literature report of syntheses of 8c and its geranyl homolog, the isolated products were invariably found mixed with 9 (8c:9~ 83:17) even after mild vacuum distillation conditions [43,44]. It should be noted the names ‘stylotellane A’ and ‘stylotellane B’ were conferred upon the carbonimidic dichloride 10, and the related 11 [10], from Styletta aurantium [45]. The latter natural product was originally isolated by Wratten and Faulkner from Pseudaxinyssa pitys – the first example of this functional group in a natural product.[10] – along with its chloro-axinyssane analog, 12. Compounds 8b,d and 10 complete the set of known farnesyl N-derivatives. It appears that 2m is the only allylic TC among these marine natural products, with the NCS group substituted at the ‘tail’ of the first isoprene group in the precursor FPP. The unexpected, unbranched long-chain lipids, 13, from Pseudoaxinyssa, containing vinyl-substituted α,ω-bisisocyanato groups, it can be noted, appear to have a different, indeterminate, biosynthesis [46].

One consequence of a strictly unimolecular reaction of NCS^– with R⁺ when the product creates a new asymmetric center at the electrophilic C, are diastereomeric mixtures. The product distribution will be dependent upon the usual factors that govern stereofacial preferences of S_N1 reactions, e.g. steric hindrance and stereoelectronic factors. For example, in the case of epimeric 2g and 2h, isolated from the sponge Axinyssa aculeata [20], the reported ratio is 3:2 [47], but from local symmetry considerations the two epimers should be about equal in terms of G˚.

When thought of in this way, capture of carbocations by free NCS^–, is a ‘clock reaction’; a kinetic monitor of the partition of the shunt reaction of cyanoformate, CF (NC-COOH Scheme 1)– dissociation to NC^– and CO₂ – and direct nucleophilic S_N1 capture by NCS^– when the ratios of epimers can be measured. Garson [11,41,48] and others [49] have shown through numerous radiolabeling experiments that inorganic [¹⁴C]CN^– is assimilated into sponge TIs. For example, 7,20-diisocyanoadociane (3) is radiolabeled by [¹⁴C]CN^– [50]. These observations have recently been interpreted as an interception of an equilibrium cyanide pool formed by dissociation of CF, the putative ‘NC’ vector, and exogenous uptaken HCN [9]. In the same study, Garson found no radiolabel incorporation into 3 when the sponge was incubated with [2-¹⁴C] Gly or a number of other amino acids [51,52].

The prevailing belief up to now was that inorganic NC^– is the precursor of TIs, but for several reasons this was shown to be untenable [9]. If it were so, a simple test can be made: the expected labeling of living sponges with [¹⁴C]-CN^– should also induce formation of limiting amounts of R-SCN from rhodanese-promoted conversion to NCS^– and the kinetic-product distribution of nucleophilic capture, but this has not been observed to date [53]. Garson and coworkers showed that radiolabeled thiocyanate (K[¹⁴N]NCS) is assimilated by living sponges into TIs including axisonitrile-3 (notably with a formula lacking S) and two ITCs (axisothiocyanate-3) albeit with far lower specific radioactivities than the same radiolabeled natural products obtained from incubations with Na{¹⁴C]CN [54]. Simultaneous labeling of axisonitrile-3 from incubation of the sponge Acanthella cavernosa [55] with K[¹⁴N]NCS requires, at the very least, an unspecified interchange of thiocyanate with K[¹⁴C]CN [56]. In a separate study, Simpson and Garson found incorporation of both [¹⁴C]SCN^– and [¹⁴C]CN^– into the sesquiterpene thiocyanato group of 2b[18,19], from Axinyssa sp. n. from the Great Barrier Reef [57]; both results are consistent with the adventitious thiocyanate model for TC biosynthesis (Figure 4, Scheme 1)

What could be testable in the field is incorporation of N¹⁴C-COOH in sponges producing TIs, ITCs and TCs or N¹⁴CS^– under more controlled conditions. The logistics of this proposed experiment are beyond the scope of this report, but the outcomes would, nevertheless, be compelling.

3. Materials and Methods

3.1. General Experimental Procedures.

DFT Calculations. All DFT calculations were performed using Spartan ’20 (Wavefunction, Irvine, CA) using functional ωB97X-D and basis set 6-31G* (H₂O or gas phase). Coordinates for the optimized structures and bond parameters can be found in the Supporting Information. See Supporting Information for complete citation to the DFT product.

4. Conclusions

A proposal is forwarded for the likely origins of the rare sponge-derived terpenoid thiocyanates (TCs) that are decoupled from the biosynthesis of isothiocyanates (ITCs). Isothiocyanates (ITCs) likely arise from a low-valent sulfur-transfer reaction to precursor terpenoid isonitriles (TIs). Adventitious thiocyanate ion, NCS^–, a competent amphiphilic nucleophile generated by rhodanese, is solely responsible for sponge TC terpenoid natural products in two ways; S_N1 addition at S produces TCs. Addition of rhodanese-derived NCS^–, at N to the incipient terpenyl carbocation R⁺, contributes to the ITC pool by simultaneous converse S_N1 reaction.