A History of Skunk Defensive Secretion ResearchWilliam F. Wood, Department of Chemistry Humboldt State University, Arcata, CA 95521 E-Mail - email@example.com
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How to deodorize skunk spray
There are five species of skunks found in North America: the striped skunk Mephitis mephitis, the hooded skunk M. macroura., the spotted skunk Spilogale putorius and S. gracilis and the hog-nosed skunk Conepatus mesoleucus *. (Spilogale putorius has recently been divided into two species, S. putorius in the Eastern part of the United States and S. gracilis in the West.) All are known for their potent means of chemical defense: the spraying of a repulsive-smelling liquid from their anal glands. For the most part, research on this secretion have been focused on the most common member of this group, the striped skunk. In this review, the term skunk refers to this species, unless otherwise specified.
Skunks and their defensive secretion have both fascinated and repelled natural products chemists. The chemicals secreted by the members of the mephatinae, a New World subfamily of the weasel family (Mustelidae) are so obnoxious that few chemists have been willing to work with them. On the other hand, once researchers publish the identity of these components, they were cited far and wide in the popular and chemical literature. This has lead to several incorrect structural identifications persisting in the literature for years since few chemists have been interested in reinvestigating these secretions.
Skunk defensive secretion has been called by many different names. A common term that has been used to describe this secretion is "skunk musk." Musk and musky are terms best used to describe the odor of the large ring ketones, muscone (I) and civetone (II), from the musk deer (Ruzicka 1926a, 1926b) and the African civet (Ruzicka 1926c, Ruzicka et al. 1927). These compounds and other similar synthetic musks are used a base odor in many perfumes and so it seems inappropriate to use the term "skunk musk" for this secretion. Better terms are "skunk spray," which describes the ejection of the secretion and "defensive secretion" which describes its natural function.
Skunks are the best known example of defensive secretions used to deter predation. This secretion is stored in two glands (anal sac) leading to nipples that are situated just outside the anal opening. When attacked or surprised a skunk will eject this secretion up to a distance of about 3 meters. At high concentrations it will cause nausea and retching in humans and like tear gas, has lachrymatorial action if it comes in contact with the eyes. As lower concentration it is highly repellant and can be detected by humans at extremely low concentrations. Aldrich (1896) showed it can be detected at 10 part per billion (10 ppb).
The first report in the chemical literature on skunk spray was by a Dr. Swarts working with Wöhler in Germany (Swarts, 1862). They had obtained a sample of the secretion form a friend in New York. Swarts analyzed the yellow oil and found it consisted of a colorless fraction boiling between 105°C and 110°C, a higher boiling yellow fraction boiling between 195°C and 200°C and a nitrogenous basic compound in the residue after distillation. In another experiment he steam distilled the secretion and found the water insoluble part of the distillate to be rich in sulfur.
Another German, Dr. O. Löw next reported work on skunk spray (1879). He confirmed Swarts' findings that it contained sulphur compounds and a nitrogenous base. His major problem in completing the research was not his chemical expertise, but the reaction of his companions and coworkers. This is clearly illustrated in a letter from Dr. Abel to Thomas Aldrich of Johns Hopkins (1896).
"On an expedition through Texas in 1872 I had frequent opportunity to collect a sufficient quantity of this secretion to establish its chemical constitution, but all my companions protested against it, declaring the odour which clung to me to be unbearable. On my return to New York City I started a few chemical tests, with the little I had collected, when the whole college rose in revolt, shouting, 'A skunk, a skunk is here!' I had to abandon the investigation."
Thomas Aldrich working in the Laboratory for Physiological Chemistry at Johns Hopkins University next investigated skunk secretion (Aldrich 1986). He had a plentiful supply of anal sacs from sources in Maine. The amount of secretion in these glands varied with a maximum of about 5 mL. He description of the secretion follows: "the secretion is a a clear, limpid fluid, of golden-yellow or light-amber colour, of a characteristic, penetrating, and most powerful odour, and having a specific gravity, at ordinary temperature, less than water (0.939)."
The belief that skunk secretion contains 1-butanethiol (III) certainly owes its source to this report. Aldrich never stated the secretion contained 1-butanethiol, however, he used 1-butanethiol as a comparison to a compound in skunk secretion to authentic samples. He did think that it was likely a major component of the secretion was an isomer of 1-butanethiol. In his second publication on this secretion (Aldrich 1897), he says this part of the secretion "is a mixture of higher mercaptans, containing among others (still undetermined) normal butyl mercaptan. The belief that skunk spray contained 1-butanethiol continued until 1975. Andersen and Bernstein (1978) review how this misconception may have been incorporated into the chemical literature.
In his first study, Aldrich distilled the secretion and found two volatile fractions, one boiling from 100-130°C and another from 130-150°C. From this he excluded several low molecular weight thiols (boiling point): methanethiol (6°C), ethanethiol (36°C), 1-propanethiol (67°C) and 2-propanethiol (57-60°C). Elemental analysis for sulfur in the low boiling fraction gives results that are close to what would be expected for 1-butanethiol or an isomer of this compound. The calculated % S for 1-butanethiol is 35.55% and Aldrich found 35.37% and 34.98%. Aldrich explained that these low values could be explained by the presence a small contamination by a higher molecular weight thiol.
Aldrich also prepared lead and mercury derivatives of the thiol in the low boiling fraction and again found the elemental analysis for carbon and hydrogen to be close to what he found for authentic 1-butanethiol derivatives of these metals. In this report he states that, "The results of these analyses are sufficiently near the theoretical figures when considered with the boiling point to convince, I think, the most sceptical that the greater part of this fraction contains one of the butyl mercaptans." The boiling point of 1-butanethiol is 97°C and the compound he isolated boiled from 100-110°C. Aldrich assumed this difference was due to the presence of higher boiling compounds in the secretion. There are three other thiols that that have the same molecular formula as 1-butanethiol: 2-butanethiol, 2-methyl-1-propanethiol and 2-methyl-2-propanethiol. Of these compounds, Aldrich attempted to synthesize 2-methyl-1-propanethiol, but did not succeed.
In his explanation of the difference in elemental analysis of his compound from the skunk and similar derivatives of 1-butanethiol, Aldrich never indicated that he considered compounds with double bonds or rings to be a possibility. After Andersen and Bernstein (1975) identified this compounds as the unsaturated, (E)-2-buten-1-thiol they reexamined Aldrich's elemental analysis (Andersen and Bernstein, 1978). For many of Aldrich's derivatives, the percentages of carbon and hydrogen are within the experimental values expected for (E)-2-buten-1-thiol.
In the following year, Aldrich published a second report on skunk defensive secretion (Aldrich and Walter 1897). In this study they identified 2-methylquinoline (IV), the nitrogenous base that had previously been reported by Swarts and Löw. Aldrich based his identification on a number of derivatives he prepared from the skunk's compound compared to a synthetic sample of 2-methylquinoline he had prepared. This compound was reconfirmed in later studies by Andersen, et al. (1982) and Wood (1990).
Stevens (1945) made a brief foray into the field of skunk research. He was looking for a source of new natural musks like civetone or muscone (see above) to be used in perfumes. He failed to find any large ring ketones but did isolate bis[2-butenyl] sulfide (V). Later work could not confirm this as a part of fresh secretion (Wood 1990) so it may be an artifact of the isolation procedure. Stevens noted the secretion was "repulsive in odor" and this may have kept him from actively working on it. The experimental procedure indicates after collection, the native secretion separated into two layers after several months of storage. Next the sample was treated mercuric chloride to remove the thiols for several days. After removal of the mercury salts, the solution was stored for "considerable time" before the solvent was removed. Vacuum distillation was used to isolate the bis[2-butenyl] sulfide.
Seventy nine years after the work of Aldrich that lead to the erroneous belief that skunks produced 1-butanethiol, Kenneth Andersen and David Bernstein at the University of New Hampshire showed this compound had been incorrectly identified (1975). The thiols they identified responsible for the stench of skunk spray were (E)-2-butene-1-thiol (VI) and 3-methyl-1-butanethiol (VII). Interestingly, Aldrich (1896) had prepared 3-methyl-1-butanethiol as part of his first study on skunk spray and speculated it may have been a reason for the faulty elemental analysis. He said his synthetic compound had the same boiling point, appearance and odor of similar fraction of skunk secretion. Alas, he never made a positive identification of this compound. The third major compound Anderson and Bernstein identified in the secretion was identified as (E)-2-butenyl methyl disulfide (VIII) on the basis gas chromatographic retention times and nuclear magnetic resonance (NMR) spectroscopy. This latter compound was later shown to have been assigned the wrong structure (Wood 1990).
In 1982 Andersen and coworkers continued research on skunk defensive secretion using gas chromatography-mass spectrometry (GC-MS). In this study mass spectra confirmed the identification of (E)-2-butene-1-thiol and 3-methyl-1-butanethiol, but were unable to find the third major component of their first study, (E)-2-butenyl methyl disulfide. Aldrich's identification of 2-methylquinoline was corroborated and a new compound, 3-methylbutanyl thioacetate (IX), was identified. Another new identification was, (E)-2-butenyl propyl sulfide (X), but unfortunately it was later shown to have the wrong structural assignment (Wood 1990).
I was the next player on skunk spray research (Wood 1990). Originally I investigated the striped skunk as a source of authentic compounds for research on the spotted skunk. Gas chromatographic-mass spectrometric analysis of fresh secretion showed differences from that of Andersen and coworkers. I prepared (E)-2-butenyl methyl disulfide and (E)-2-butenyl propyl sulfide, compounds previously reported from the secretion, but failed to find them in the secretion. Also, the third most abundant compound in my analysis of fresh secretion appeared to be previously unreported. This compound had the same molecular weight (molecular ion) as(E)-2-butenyl propyl sulfide the compound identified in Andersen's second study, but it has a different mass spectral fragmentation pattern. A possible candidate compound with this molecular weight, S-(E)-2-butenyl thioacetate (XI) was prepared. This compound had identical properties by GC-MS analysis to the compound in the secretion and also explains the disappearance of (E)-2-butenyl methyl disulfide from Andersen's second analysis. Thus it is likely that (E)-2-butenyl methyl disulfide and (E)-2-butenyl propyl sulfide reported by Andersen are in reality S-(E)-2-butenyl thioacetate.
The incorrect assignment of these structures is rather easy to explain. (E)-2-Butenyl methyl disulfide and the new compound S-(E)-2-butenyl thioacetate have almost identical NMR spectra and gas chromatographic retention times. Identical NMR spectra and gas chromatographic retention times are usually all that is needed to assign a new structure, so Andersen and Bernstein had no reason to perform further tests. Also, the gas chromatographic retention times of S-(E)-2-butenyl thioacetate and (E)-2-butenyl propyl sulfide are almost identical.
In this study, I identified several new compounds and verified many from previous studies. New compounds identified in this study were S-(E)-2-butenyl thioacetate, 2-quinolinemethanethiol (XII) and S-2-quinolinemethyl thioacetate (XIII). The previously identified 2-methylquinoline (Aldrich and Jones 1897), (E)-2-butene-1-thiol, 3-methyl-1-butanethiol and 3-methylbutanyl thioacetate (Andersen and Bernstein 1975) were also seen in this study.
To sumerize, this latest research shows striped skunk spray to have 7 volatile components greater than 1%. Three are thiols, three are thioacetate derivatives of these thiols and the final compound is the alkaloid 2-methylquinoline. The structures of these thiols and thioacetates follows. The amounts are the variation found in 4 skunks.
The Spotted Skunk (Spilogale gracilis)
(Spilogale putorius has recently been divided into two species, S. putorius in the Eastern part of the United States and S. gracilis in the West.)
After I finished the work on the striped skunk I returned to my study of the spotted skunk. This work was made easier by the striped skunk research. The defensive secretion of the spotted skunk differs from that of the striped skunk in that it only contains thiols and none of the thioacetates found in striped skunk secretion (Wood et al. 1991). The two major thiols of the striped skunk, (E)-2-butene-1-thiol and 3-methyl-1-butanethiol are also the major components in the secretion of the spotted skunk. A third thiol, 2-phenylethanethiol (XIV), was present at moderate concentration in this smaller skunk. (A number of minor compounds were identified from this species. They are detailed in on Chemistry of Skunk Spray that can be accessed from the skunk spray page.)
Hog-nosed Skunk (Conepatus mesoleucus*)
An Argentinian species of the hog-nosed skunk (Conepatus suffocans) was investigated by Fester and Bertuzzi (1937). These researchers had only 4 mL of secretion and were able to isolate 0.42 g of a yellow liquid with a mercaptan-like odor. From this small quantity, they were not able to make a positive identification of this compound, but did state it was very similar but not identical to 1-butanethiol.
Analysis of this secretion from a North American hog-nosed skunk differs from that of the spotted skunk and the striped skunk (Wood et al. 1993). Like the striped skunk, it does contain thioacetate derivatives of the thiols in the secretion. Also, a major component of the striped and spotted skunks' secretion is missing from the secretion of this species. The major components from this skunk's secretion are (E)-2-butene-1-thiol and S-(E)-2-butenyl thioacetate. (A number of minor compounds were identified from this species. They are detailed in on Chemistry of Skunk Spray that can be accessed from the skunk spray page.)
Since the hog-nosed skunk's secretion contains a thioacetate derivative of the major thiol and does not contain any 2-phenylethanethiol, it is more like the secretion of the striped than spotted skunk. This may indicate the hog-nosed skunk and striped skunk are more closely related to each other than either is to the spotted skunk. A comparison of the major volatile components from these three species follows (Wood, 1993).
A comparison of major volatile components of anal sac secretion from three species of North American skunks. (ND = none detected, Range data from 4 striped skunks, 2 spotted skunks and one hog-nosed skunk)
|S - (E )-2-butenyl thioacetate||
|S -3-methylbutanyl thioacetate||
|S -2-quinolinemethyl thioacetate||
Glossary of Names and Terms
The nomenclature of chemicals in this review use current rules codified by the International Union of Pure and Applied Chemistry (IUPAC). Much of the older literature uses a different nomenclature systems reflecting usage at that time. A major shift in chemical names has been to replace the term mercaptan by thiol for the R-SH functional group. Designation of the spacial orientation of groups attached to a double bond has also changed. The terms cis and trans have been replaced by the prefixes (E) and (Z). These terms use different rule to determine each type of prefix so there is no way to directly translate the use of these prefixes from one system to another. However, in many cases the use of the prefix trans will coincide with the prefix (E)-.
List of names used in this review and names used in previous reports.
1-butanethiol = butyl mercaptan, normal-butyl mercaptan, n-butyl mercaptan
(E)-2-butene-1-thiol = trans-2-butene-1-thiol, crotyl mercaptan
3-methyl-1-butanethiol = iso-amyl mercaptan, isoamyl mercaptan
2-methylquinoline = alpha-methyl-quinoline
(E)-2-butenyl methyl disulfide = trans-2-butenyl methyl disulfide
(E)-2-butenyl propyl sulfide = trans-2-butenyl propyl sulfide
Acknowledgements: I thank Professor Kenneth K. Andersen for copies of the report on Löw's work and a translation of Fester and Bertuzzi's study. This page is an adaptation of a seminar presented to the Chemistry Department at Humboldt State University.
*Recent research indicates the two species of hog-nosed skunks, Conepatus mesoleucas and C. leuconotus may be the same species. Thus, according to the rules of zoological nomenclature, the one that was described first has priority. If they are the same species, the name C. leuconotus will be used. (Pers. Communication Dr. Jerry Dragoo, University of New Mexico, firstname.lastname@example.org)
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©William F. Wood 1998. Updated on 6 October 1998.