{"id":10081,"date":"2016-08-23T12:54:34","date_gmt":"2016-08-23T16:54:34","guid":{"rendered":"https:\/\/www.masterorganicchemistry.com\/?p=10081"},"modified":"2022-10-31T06:06:11","modified_gmt":"2022-10-31T11:06:11","slug":"structure-determination-case-study-deer-tarsal-gland-pheremone","status":"publish","type":"post","link":"https:\/\/www.masterorganicchemistry.com\/2016\/08\/23\/structure-determination-case-study-deer-tarsal-gland-pheremone\/","title":{"rendered":"Structure Determination Case Study: Deer Tarsal Gland Pheromone"},"content":{"rendered":"

A Roadmap For Structure Determination of Organic Compounds<\/strong><\/p>\n

Male black-tailed deer emit a pheromone from their tarsal gland that attracts female deer.<\/p>\n

Say you had a bunch of deer tarsal glands available and wanted to try to identify the active molecule from that gland.<\/p>\n

How would you go about identifying the structure of the pheromone? What techniques would you use? How do you go from an “unknown” mixture to a “known” molecule?\u00a0<\/strong>That’s what this post is about.<\/p>\n

Table of Contents:<\/strong><\/p>\n

    \n
  1. The\u00a0 Four Stages of Natural Product Isolation<\/a><\/li>\n
  2. Natural Product Isolation\u00a0 Case Study: Deer Tarsal Gland Pheromone<\/a><\/li>\n
  3. A Roadmap for Structure Determination<\/a><\/li>\n
  4. Analysis of an Unknown Compound<\/a><\/li>\n
  5. Proposing a\u00a0 Structure Based On The\u00a0 Evidence<\/a><\/li>\n
  6. Conclusion: Structure Determination\u00a0<\/a><\/li>\n
  7. Notes<\/a><\/li>\n<\/ol>\n
    \n

    <\/a>1. The 4 Stages Of Natural Product Isolation<\/h2>\n

    In January 1969, the Beatles released their “Yellow Submarine” single and performed their last live gig<\/a>, Richard Nixon was inaugurated as president, and Brownlee et. al.\u00a0published their seminal paper, “Isolation, Identification, and Function of the Chief Component of the Male Tarsal Scent in Black-tailed Deer<\/a>” in the scientific journal Nature<\/a>.<\/p>\n

    It’s that last part that we’re going to focus on\u00a0today.<\/p>\n

    \"screen<\/sub><\/p>\n

    *statement may not be completely accurate<\/sub><\/p>\n

    The Beatles Rooftop Concert 1969 London (HD)<\/p>\n

    Doesn’t Ringo look miserable?<\/p>\n

    Now on to something even more exciting: natural product isolation, which we’ve been going through over the past few posts.<\/p>\n

    You can\u00a0think of the isolation of natural products (molecules) as having\u00a0four main stages.<\/p>\n

    \"4<\/p>\n

    Stage 1: Curiosity<\/strong>. What makes “hot peppers” hot? What’s responsible for the painkilling properties of opium? How do insects find each other to mate, even from miles away? What gives vanilla that lovely aroma? [We went through some interesting molecules from nature in this post<\/a>.]<\/p>\n

    Stage 2: Extraction<\/strong>. Following our curiosity, we need to find a way to get the molecules of interest out of the organism and into a flask. “Extraction” of the organism with an appropriate solvent is usually how we do it, which we covered here<\/a>.<\/p>\n

    Stage 3: Purification<\/strong>. \u00a0How can we separate a crude mixture into its components, so that we can determine which molecular species are present? We covered that in the last post, here<\/a>.<\/p>\n

    Stage 4: Analysis.<\/strong> How do we determine the structure of a pure, unknown compound? This is what we’ll talk about today, and for a good long while in the future, because it’s not<\/em>\u00a0a small topic.<\/p>\n

    <\/a>2.A Natural Product Isolation Case Study: Isolation and Analysis of Deer Tarsal Gland\u00a0<\/strong>Pheromone<\/b><\/h2>\n

    Since we’ll be covering analysis for a long time, why not start with something fun? In this post we’ll present a case study on how Brownlee et. al. began with an observation and a question (“Black-tailed deer smell each other’s tarsal areas\u00a0a lot. What molecules are in the tarsal gland?”) and followed it through to determine the structure of the key pheromone below, which is an important signalling molecule for male black-tailed deer.<\/p>\n

    \"sniffing<\/p>\n

    This study nicely illustrates the analysis of an unknown natural product.\u00a0\u00a0It\u2019s also fairly simple, and you don\u2019t need any knowledge of NMR [which we\u2019ll cover in more detail later] to follow the logic of how the structure was obtained.<\/p>\n

    1. ‘Curiosity’ .\u00a0<\/em>Male deer of the genus\u00a0Odocoileus<\/em> contain a tarsal organ on their ankle which consists of scent glands and is covered with long tufts of bright, stiff hairs. Members of an established herd check each other’s tarsal tuft occasionally (once or twice an hour for each individual). When a strange female or strange male approaches the herd, the frequency of smelling tarsal turfs increases\u00a0up to\u00a06-16 times an hour (for a strange male). Fawns recognize their mother by sniffing her tarsal organ. When threatening each other, bucks often urinate on the tarsal tufts and rub them together simultaneously.<\/p>\n

    Clearly there’s a key molecule (or molecules) that provides this chemical signal. What’s its structure?<\/p>\n

    \"black<\/em><\/p>\n

    2. Extraction<\/em>. Tarsal organs from male black-tailed deer killed in the same area during the same week were excised. The hair tuft was rinsed with petroleum ether<\/a> (a mix of short-chain alkanes) and the collected extracts, after removal of petroleum ether, were distilled at 80-100\u00a0\u00b0C at reduced pressure to give a crude oil.<\/p>\n

    3. Separation.\u00a0<\/em>The extract was analyzed with gas chromatography (GC) which showed that the tarsal gland has many<\/em> chemical components.\u00a0\u00a0 The numbers at the bottom are in minutes: the major component (labelled 66) from the male tarsal gland at 16 minutes was chosen was chosen to be investigated. (Interestingly, the fraction labelled 26 at about 6 minutes was found to be the major component of female tarsal glands. )<\/p>\n

    \"GC<\/p>\n

    Isolation and analysis of this component was performed using “preparative” gas chromatography.\u00a0Just for fun, I’m including full conditions in Note 1<\/a><\/span>: Some of the jargon has been “decoded” to make it more accessible.<\/span><\/p>\n

    4.\u00a0<\/em>Analysis.\u00a0<\/em>Here\u2019s where we really begin<\/strong>.\u00a0The authors mention that a single buck organ yielded 10-80 micrograms <\/em>(\u03bcg). 30 micrograms were used for analysis. That’s 1\/30 of a milligram, folks: extremely impressive even by today’s standards.<\/p>\n

    Before diving in, let’s take a moment to survey the landscape.<\/p>\n

    Let’s say\u00a0you have a pure sample of an unknown compound. Where do you start? What are the most important questions to ask?<\/p>\n

    <\/a>3.A Roadmap For Structure Determination<\/strong><\/h2>\n

    I might humbly suggest the following, with the most important questions listed first.<\/p>\n