Synthesis of 5-(3,4-difluorophenethoxy)-3-(4-(pyrrolidin-1-yl)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine


VTM010 (0.05 g, 0.12 mmol) was dissolved in degassed toluene (2 mL). Pyrrolidine (0.02 g, 0.288 mmol), NaO t Bu (0.016 g, 0.168 mmol), JohnPhos (1.6 mg, 5.4 μmol) and Pd 2 (dba) 2 (0.3 mg, 0.6 μmol) were mixed in this order and stirred for two weeks at 100°C. The reaction was quenched (2M HCl). The organic layer was extracted (3x2mL EtOAc). The crude was concentrated in vacuo and purified via column chromatography (EtOAc).


NMR analysis suggested that a final product was not formed.

Synthesis of 3-(4-bromophenyl)-5-(3,4-difluorophenethyl) – [1,2,3]triazolo[4,3-a]pyrazine



VTM009 (0.738 g, 1.22 mmol) was added to a solution of 3,4-difluorophenyl-1-ol (0.60 g, 4.16 mmol) in dry THF (30 mL). The mixture was cooled down to 0°C and NaH (60%, 0.17 g, 6.93 mmol)was added. The mixture was stirred at room temperature for 2 hours. The reaction was quenched with HCl (30mL) and the organic layer extracted with EtOAc (3x30 mL) and 2M HCl (3x10 mL). The crude was concentrated under reduced pressure

VTM010 CRUDE (2).jpg

and purified via column chromatography (100% EtOAc) to yield the final product (0.50 g, 95%) as a beige solid.

VTM010 PRODUCT (3).jpg

Tissue Sampling – Crassostrea virginica Tissues for Archiving

I figured it’d be prudent to collect some Eastern oyster (Crassotrea virginica) to have around the lab.

I used one of the C.virginica oysters that I picked up Taylor on 20171210 for sampling.


  • Upper mantle (avoided area that was near gonad/white-ish)
  • Ctenidia
  • Lower mantle
  • Muscle
  • Gonad

Samples were transferred to 1.7mL snap cap tubes, frozen on dry ice, and stored @ -80C in Rack 13, Col 1, Row 5.

Software Install – MSMTP For Email Notices of Bash Job Completion on Emu (Ubuntu)

After I finally resolved the installation of PB Jelly on Emu (running Ubuntu 16.04), I’ve had a PB Jelly assembly running for the past two weeks! I’ve gotten tired of checking on its status (i.e. is it still running?) every day, so I dove in and figured out how to set up Emu to email me when the job is complete!

To get this going, I mainly followed this msmtp ArchWiki guide., but here are the specifics of how I set it up.

Step 1. Installed a mail server:

sudo apt-get install sendmail

Step 2. Installed msmtp:

sudo apt-get install msmtp

Step 3. Created the following file in my home directory (/home/sam/): ~/.msmtprc

The original contents of the file for testing were:

       # Example for a user configuration file ~/.msmtprc
       # This file focuses on TLS and authentication. Features not used here include
       # logging, timeouts, SOCKS proxies, TLS parameters, Delivery Status Notification
       # (DSN) settings, and more.

       # Set default values for all following accounts.

       # Use the mail submission port 587 instead of the SMTP port 25.
       port 587

       # Always use STARTTLS.
       tls on
       tls_starttls on
       tls_certcheck off
       # A freemail service
       account uw

       # Host name of the SMTP server

       # Envelope-from address

       # Authentication. The password is given using one of five methods, see below.
       auth on
       user samwhite

       # Password method 3: Store the password directly in this file. Usually it is not
       # a good idea to store passwords in plain text files. If you do it anyway, at
       # least make sure that this file can only be read by yourself.
       password myuwpassword

       account default : uw

This is a configuration to allow emails to get sent via the Univ. of Washington email servers. Yes, I currently had UW password saved in this file, but will be addressing this issue below.

Step 4. Changed permissions on ~/.msmtprc to be readable/writable only by me (important, particularly if you’ve stored your password in this file!):

chmod 600 ~/.msmtprc

Step 5. Assigned sendmail to use msmtp with the set command (this sets the following command as a positional parameter by adding to the /etc/mail.rc file:

echo "set sendmail=/usr/bin/msmtp" | sudo tee -a /etc/mail.rc

This command pipers the output of echo to sudo and uses tee -a to append to our desired file (/etc/mail.rc).

Step 5. Send a test email:

echo "Job complete!" | msmtp

That will send an email with no subject and the body of the email will contain “Job complete!”.

That’s the basic set up for this.

To use it in your workflow, you’d append that command to the end of any Bash command or in a separate Jupyter notebook cell that is queued to run after a previous cell completes it’s job.


echo "This counts as a command"; echo "Job complete!" | msmtp

This will run the first echo command. When that finishes, then the email command will run. You can get fancy and have different emails in response to how the running program exits (i.e. fails or is successful) and send different email responses, but I’m not going to get into that.

Anyway, not bad! However, we want to make this a bit nicer and more secure.

Improve security:

Step 1. Generate a GPG Key:

Follow the instructions under the Creating an Encryption Key section at this link.


Technically, this is does not follow proper security protocols, but this is better than having a plain text password, and setting it up this way is the only way the mail program will send without prompting the user for a password (which kills the automation we’re trying to achieve).

Step 2. Create an encrypted password file:

gpg --encrypt -o ~/.msmtp-password.gpg -r youremailaddress -

After entering that, type your UW email password(NOTE: You will not receive a new prompt, so just type it in), and then Enter. Then, press Ctrl-d.

Step 3. Add the following line to your ~/.msmtprc file:

passwordeval    "gpg --quiet --for-your-eyes-only --no-tty --decrypt ~/.msmtp-password.gpg"

Here’s what the file looks like now:

       # Example for a user configuration file ~/.msmtprc
       # This file focuses on TLS and authentication. Features not used here include
       # logging, timeouts, SOCKS proxies, TLS parameters, Delivery Status Notification
       # (DSN) settings, and more.

       # Set default values for all following accounts.

       # Use the mail submission port 587 instead of the SMTP port 25.
       port 587

       # Always use STARTTLS.
       tls on
       tls_starttls on
       tls_certcheck off

       # Email account nickname
       account uw

       # Host name of the SMTP server

       # Envelope-from address

       # Authentication. The password is given using one of five methods, see below.
       auth on
       user samwhite

       # Password method 2: Store the password in an encrypted file, and tell msmtp
       # which command to use to decrypt it. This is usually used with GnuPG, as in
       # this example. Usually gpg-agent will ask once for the decryption password.
       passwordeval    "gpg --quiet --for-your-eyes-only --no-tty --decrypt ~/.msmtp-password.gpg"

       account default : uw

Step 4. Change permissions on ~/.msmtp-password.gpg so it’s only readable/writable by you:

chmod 600 ~/.msmtp-password.gpg

Step 5. Send a test email like before:

echo "Job complete!" | msmtp

That’s it for security.

Add a subject to the emails:

Step 1. Create ~/.default_subject.mail and add the following lines to the file (substitute your own email address):

From: [EMU]

Feel free to change the Subject and/or From info to whatever you’d like.

Step 2. Send message using ~/.default_subject.mail:

cat ~/.default_subject.mail | msmtp

To use this in your workflow, you’ll do just like before (but using the command immediately above) and append to the end of any Bash command.

Make it short & sweet

Appending those lines is going to be difficult to remember, is annoying to type out, and displays your email address (particularly if using a publicly hosted Jupyter notebook like most of us in lab do). Here’s a nice way to remedy that.

Step 1. Add email address as variable in ~/.bashrc:

Add the following lines to the end of your ~/.bashrc file:

# Email address

Your email address is now saved in the variable $EMAIL. You will need to use the following command to load that information:

source ~/.bashrc

Verify that it worked:

echo "$EMAIL"

That should spit out your email address and is ready to be used!

Step 2. Add alias for full mail command to ~/.bash_aliases file:

echo "alias emailme='cat ~/.default_subject.mail | msmtp "$EMAIL"'" >> ~/.bash_aliases

Verify that it worked:

source ~/.bash_aliases

So, from now on, all you have to do is append the command emailme to the end of any Bash commands and you’ll get email when the job is finished!!! You can edit Steps 1 & 2 to use a variable other than “EMAIL” and an alias other than “emailme” – use whatever you’d like.

DNA Sonication & Bioanalzyer – C. virginica gDNA for MeDIP

I transferred 8ug (136uL) of Crassotrea virginica gDNA (isolated earlier today) to two separate 1.7mL snap cap tubes for sonication/shearing.

I performed shearing at the NOAA Northwest Fisheries Science Center, using the Qsonica Q800R. Mackenzie Gavery assisted me.

Target fragment size was ~500bp.

Samples were run at the same time with the following settings:

  • 10 minutes
  • 30 seconds on, 30 seconds off
  • 25% power

After sonication, fragmentation was assessed using the Seeb Lab’s Bioanlyzer 2100 (Agilent) and the DNA 12000 Chip Kit (Agilent). NOTE: All of the reagents and the chips were past their expiration dates (most in June 2016).


Agilent 2100 Bioanalyzer Expert file (XAD): 2100 expert_DNA 12000_DE72902486_2017-12-11_13-45-31.xad

Fragmentation was successful, and pretty consistent.

Both samples appear to have an average fragment size of ~420bp. Will proceed with MeDIP, once reagents are received.

Unsheared gDNA:

DNA Isolation & Quantification – Crassostrea virginica Mantle gDNA

DNA was isolated from a single adult Eastern oyster (Crassostrea virginica) for a pilot project with Qiagen to test their new DNA bisulfite conversion kit. The oyster was obtained yesterday afternoon (20171210) from the Taylo rShellfish Pioneer Square location. The oyster was stored @ 4C O/N.

The oyster was shucked and four pieces of upper mantle tissue (~35mg each) were snap frozen in liquid nitrogen (LN2). Tissues were pulverized under LN2 and then DNA was isolated separately from each sample using the E.Z.N.A. Mollusc DNA Kit (Omega) according to the manufcaturer’s protocol.

Samples were eluted with 100uL of Elution Buffer and were pooled into a single tube.

The gDNA was quantified using the Qubit 3.0 (Invitrogen) and Qubit dsDNA Broad Range Kit (Invitrogen), using 5uL of sample.


Qubit (Google Sheet): 20171211_qubit_virginica_DNA

Concentration is 58.4ng/uL.

That makes the total yield ~23.36ug (23360ng). This is more than enough to perform two separate MeDIP preps and two separate reduced representation digestions with MspI.

Will proceed with shearing of DNA for MeDIP.

Attempted synthesis of final product using Buchwald-Hartwig cross-coupling on a larger scale


Synthesis of final product using Buchwald-Hartwig cross-coupling - on a larger scale



mechanism final step.jpg
Compound Data(mr/density) 


 mMol Eqv 
NH1-007 412.9  0.100g  0.24 
Toluene 4.00ml  -
Pyrrollidine  71.2/0.85g/ml  0.048ml 0.28 1.2 
NaOtBu 96.1  0.0323g  0.34  1.4 
Pd(dBA)2  575.00 0.00069g  0.0012  0.50% 
Johns Phos 298.40 0.00322g 0.0108 4.5%

Procedure- NH1-008

NH1-007 (0.100g, 0.24mmol) was dissolved in degassed toluene (4.00mL). Pyrrolidine (0.048mL,0.28mmol) was added dropwise. John Phos (0.00322g, 0.0108mmol) was added followed by the base, NaOtBu (0.0323g, 0.34mmol). Pd(dBA)2 (0.00069g, 0.0012mmol) was added and the reaction was left to stir at 80 degrees for 7 days.

NH1-008 was separated using HCl and EtOAc. This was then dried using MgSO4, filtered and dried in vacuo. TLC was carried out (5% MeOH/95% DCM) and is shown below.

final TLC.jpg

1H NMR of NH1-008 was taken in chloroform. The NMR did not show the significant product peaks. To confirm this, NH1-008 was purified via flash column chromatography (2% MeOH/98% DCM). The TLC concluded a potential product in vials from 9-16. These were collected and dried in vacuo. 1HNMR was taken but unfortunately the analysis concluded that this reaction did not work as the spectra was same as the starting material.

Repeated SnAr Reaction


SNAr reaction 


week 3.jpg

Reagent Equivalent Molecular Weight mMol Quantity
4-flurophenylethan-1-ol 2 140.00 7.10 0.9mL
Sodium Hydride 2 24.00 14.06 540.00mg
Triazolopyrazine 1 309.55 3.55 1100.00mg
THF - - - 30.00mL


THF (30.00mL) was added to Triazolopyrazine (1100.00mg, 3.5mmol) and the mixture was stirred under a nitrogen atmosphere. 4-flurophenylethan-1-ol (0.9mL, 7.10mmol) was added and the mixture was cooled in ice. NaH (540.00mg, 14.06mmol) was added and the dark brown mixture was stirred and left under a nitrogen atmosphere for 2 hours (RMTP).  

nitrogen atmosphere.jpg

TLC analysis (Petroleum ether/EtOAc 25:75) confirmed that the reaction had worked.

HCl (2M) and EtOAc (60.00mL) were added to separate the solution, the sample was filtered. The solution was dried over MgSO4 which was filtered off and rinsed with EtOAc. The solution was evaporated in vacuo. 

The crude was purified via flash column chromatography (100% Ethyl acetate). TLC analysis (100% ethyl acetate) showed that test tubes 19-57 contained the product.

repeat 1-15.jpg
repeat 17-32.jpg
repeat 33-47.jpg
repeat final 57.jpg

The product was collected and dried in vacuo. This formed golden crystals.

1H NMR of the product NH-1-007 was taken in chloroform. The NMR showed the desired structure.

The product was weighed and dissolved in CDCl3 to ensure that no product was left in the round bottom flask. This was left to dry. The product was re-weighed (360.00mg, 0.87mol, 24%)

Analysis of product NH-1-004

δH(400 MHz, CDCl3: 9.01 (3H, s, CHAr), 7.61 (4H, d, CHAr), 6.91 (2H, d, CHAr), 6.78 (2H, d, CHAr) 4.42 (6H, s, CHAr) and 2.94 (6H, s, CHAr).